The use of sexually propagated corals is gaining popularity as an approach for reef restoration. However, manually attaching substrates with recently settled corals to the reef using binding materials is both time-consuming and expensive, limiting the use of this technique to small spatial scales. We present a novel approach whereby young corals are ‘seeded’ on the reef without the need for manual attachment to the benthos. We tested two tetrapod-shaped concrete substrates (7.9 and 9.8 cm in diameter) on which coral larvae were settled. The tetrapods were efficiently deployed by wedging them in reef crevices, in 1.5 to 7% of the time required for traditional outplanting techniques. Seeding tetrapods was most effective in reefs with moderately to highly complex topographies, where they rapidly became lodged in crevices or cemented to the benthos by encrusting organisms. After one year, average recruit survival was 9.6% and 67% of tetrapods still harboured at least one coral colony, and overall, this approach resulted in a 5 to 18 fold reduction in outplanting costs compared to common outplanting methods. This seeding approach represents a substantial reduction in costs and time required to introduce sexually propagated corals to reefs, and could possibly enable larger scale reef restoration. These measures can be ‘passive’ whereby natural recovery is facilitated through human intervention 2, or take the form of ‘active’ measures whereby humans directly manipulate the dynamics of degraded reef ecosystems 3. Because many coral reefs are assumed to no longer recover naturally from anthropogenic stressors1, active restoration approaches are increasingly considered, in conjunction with passive management interventions, to rehabilitate degraded reef communities.The loss of ecological functions and ecosystem services provided by coral reefs worldwide has prompted conservation and management efforts to promote their recovery by addressing local causes of decline4. Corals for outplanting are typically clonal asexual fragments or naturally dislodged “fragments of opportunity” of extant colonies5. Fragments are often grown-out in coral nurseries prior to outplanting and, when outplanted, have been observed to locally increase the abundance and diversity of fish6. However, the use of clonally produced fragments also results in limited genetic diversity within recipient populations, and thus may reduce their potential to adapt to changing environmental conditions7. In contrast, the use of sexually produced corals, whereby genetic recombination ensures the formation of new genetic varieties, preserves genetic variation within outplanted corals during restoration efforts4. Consequently, the use of sexually produced corals can complement more commonly used clonal approaches and provide the possibility for genetic adaptation to climate change7.Outplanting corals into degraded areas is a common active restoration approach aimed at increasing coral cover and structural complexityex situ fertilization, sexually produced coral larvae are generally settled on artificial settlement substrates (“settlement tiles”)8, that are either directly outplanted to the reef 10, or kept in land- or ocean-based nurseries13 where coral settlers are grown to sizes  that make them less vulnerable to predation and competition15. The success of sexual coral propagation techniques has improved over recent years. While large numbers of outplanted corals regularly survive past the age of one year17 and outplanted corals have reached sexual maturity in a few occasions19, mortality among newly settled corals remains extremely high 20 compared to restoration approaches using clonal fragments. Typically less than 5% of all cultured settlers survive for more than one year4, and high  levels of post-settlement mortality therefore greatly reduce the effectiveness of restoration methods using sexually produced larvae.Following gamete collection and 22. The process of outplanting artificial substrates with settled corals to the reef typically accounts for 30% of the total restoration costs when individual corals or substrates are manually secured using binding materials . In contrast, gamete collection, larval rearing and larval settlement combined typically account for less than 50% of costs4. Current outplanting techniques require tedious handling of binding materials underwater and are therefore time consuming. For example, previous studies found that between 4 and 20 min are needed to outplant a single substrate with coral settlers to the reef 13. Restoration efforts using sexually propagated corals would especially benefit from new technologies that enable cheap and fast outplanting and increased settler survival.The high costs of both asexual and sexual restoration approaches limit their application to spatial scales (<1 hectare) that are generally too small to re-establish ecological functions of degraded reef systems23. Two different tetrapod-shaped substrates were designed: Type I  relative to each other , and made of concrete. Moulds were made from polyurethane and manufactured with a multi-axis-milling machine . The tetrapods were casted in concrete between March and July 2013 and made from a homogenous mixture of 2 parts Portland cement, 4 parts river sand and 1 part water. This mixture could be easily poured in the moulds and dried rapidly. Biodegradable vegetable oil was sprayed into the moulds prior to pouring to prevent the concrete from sticking to the moulds’ sides. The concrete was allowed to dry for 24 h before the tetrapods were extracted from the moulds.30. The aquarium system consisted of five flow-through aquaria  that were continuously supplied with natural seawater (~2300 L h−1) from a nearby reef. See Chamberland et al.9 for a detailed description of this system.Experiments were conducted on Curaçao , a Caribbean island located 60 km north off Venezuela. The tetrapods (n = 80 of each type) were incubated in a flow-through seawater aquarium system for six months to wash out potentially toxic and alkaline agents from the cement mixture and allow the development of biofilms that induce coral settlement and metamorphosisFavia fragum (Esper 1797) releases planula larvae 6 to16 days after the new moon throughout the year31. Fifty adult F. fragum colonies were collected from the Curaçao Sea Aquarium reef  two days before the onset of their planulation cycle in March 2014 and kept in the aforementioned flow-through system. Between days 6 and 10 after new moon and one hour before sunset, colonies were placed overnight in two 70-L plastic cool boxes  containing ~60 L of 50-µm-filtered seawater. Every morning (between 7:00 and 8:00), all larvae released during the preceding night were collected using glass pipettes and distributed randomly among eight plastic containers  filled with ~23 L of 50-µm-filtered seawater, larvae collected during previous nights, and 10 Type I and 10 Type II tetrapods. The parent colonies were then removed from the cool boxes and returned to the flow-through system. All collected larvae were divided among the eight containers resulting in a total of ~600 coral larvae per container. Containers with coral larvae were partially submerged in the flow-through system to maintain natural seawater temperatures (28–29 °C) and water inside the containers was exchanged daily (~75%) to maintain water quality. Two airlifts were placed in the opposite corners of each container to generate water movement and prevent the formation of stagnant water in between the tetrapods. Larvae were left in the containers for five days to settle after which all tetrapods were transferred to the flow-through system.2 of available surface area per tetrapod type, surface orientation and microhabitat type. Until they were seeded to the reef, all tetrapods with ≥1 live coral settler  were hung ~50 cm below the water surface using 27.2-kg strength fishing line tied to PVC frames placed on top of the flow-through aquaria.Larval settlement rates on each tetrapod were assessed immediately before outplanting using a blue light  that causes settled larvae to fluoresce. To determine if settlement preferences differed between the two tetrapod designs , the different surface orientations  and the microhabitats types , the position of each settler on each tetrapod was mapped. Settlement rates were calculated as the number of settlers per cmF. fragum larvae had settled, SUs were seeded at the Curaçao Sea Aquarium reef, a relatively healthy reef approximately 100 meters from our rearing facility. Tetrapods were seeded within a 150 × 10 m area parallel to the coast at depths between 4 and 6 m and individually placed in a habitat of Low, Medium, or High structural complexity. These different habitat types occurred interspersed as small patches (2–10 m in width) within the outplanting area. Assignments to structural categories were made visually following Wilson et al.26. Low, Medium, or High structural complexity corresponded, respectively, to low and sparse relief, moderately complex, and very complex with numerous fissures and caves  to minimize potential confounding effects of density dependent processes29. SUs were transported from the aquaria to the reef while hanging from the same PVC frames , Type II: 0.24 (±0.08 SD), mean number of settlers per cm32  for each time point so the position of each SU could be tracked through time using natural landmarks and the tags for scale  was also quantified from photographs for each time point using ImageJ. Lastly, we calculated the proportion of outplanted SUs that could be found and still harboured ≥1 settler  through time for all treatments, henceforth referred to as ‘SU yield’. SU yield serves as a measure of success to compare the effectiveness of different restoration approaches, assuming that a single, large, coral colony can theoretically grow to adulthood per outplanted SU4.To quantify survival rates of coral settlers on each tetrapod type in the three levels of topographic complexity, the number of live Millepora spp. and Acropora palmata colonies within the study area. A total of nine tags (out of 30) could no longer be located and were likely buried under scattered Millepora and Acropora fragments or had detached. Tetrapods associated with these tags were excluded from the analysis at this time point. Dispersal distances for all tetrapods could not be measured, because most natural landmarks had also been covered or were no longer present.One week before the last survey (t = 12 months), a storm caused major breakage of 4, and compared to the few existing studies that quantified costs associated to outplanting techniques for sexually propagated corals that have used binding materials. The latter studies included restoration approaches that (1) tied other types of substrates to a rope previously nailed on the reef 9, (2) epoxied substrates to the reef 19, and (3) secured substrates in holes previously drilled in the reef framework13. Expenses associated with larval rearing  can significantly vary depending on species, rearing techniques, nursery types, and the duration of the nursery period17. This study specifically aimed at increasing the cost-effectiveness of the outplanting phase. Thus, in order to compare outplanting costs among studies, expenses related to the larval rearing phase were not considered in the cost-analysis. The analysis therefore only included expenses related to (1) the production or purchasing of settlement substrates, (2) materials needed to secure the substrates to the reef , (3) air tanks for SCUBA divers, and, if needed, pneumatic drills, and lastly, (4) labour required to carry out the outplant , and only included the time required to carry out the outplant itself. The time to prepare for dives and reach outplanting sites were not considered as the latter significantly vary among locations. The time needed for divers to wedge one SU in the reef framework was calculated from video footage taken during outplanting and was measured as the time from when a diver first held a SU in his hand ready to seed it, until the SU was wedged in the reef. The time required to outplant substrates using other outplanting techniques than seeding was taken from above-mentioned studies. To compare the total costs of the different restoration approaches, the costs to restore one hectare of reef with 10,000 SUs with 10 persons was calculated for each method, and its effectiveness expressed as SU yield after one year. Because settler mortality is highest during the first year of outplanting14, the SU yield after one year was assumed to be an adequate metric to evaluate the long-term success of sexual coral restoration efforts.Labour was expressed in terms of person-hour and converted to US dollars based on the median worldwide GDP at the time the work was carried out34 was used followed by Tukey’s post-hoc HSD tests because data did not meet the assumption of homoscedasticity. Independent replicates  per surface orientation/microhabitat type were used for the analysis . Differences in settler survival among tetrapod types, microhabitats and levels of structural complexity were compared with Kaplan-Meier’s survival analysis35 followed by log-rank (Mantel-Cox) pairwise comparisons. Fisher’s exact test of independence was used to test for differences in attachment rates of the tetrapods on the reef, as well as differences in the proportion of tetrapods still harbouring at least one coral individual through time . One-way ANOVAs were used to assess potential differences in settler growth, whereas differences in dispersal rates were tested with repeated measures ANOVAs. All analyses were performed in SPSS 24.036. Statistical values for ANOVAs and post-hoc pairwise comparisons are available as online supplementary information , surface orientations  and microhabitats , Welch’s F-test for unequal variancesAll research was carried out under the research and collecting permits granted to the CARMABI Foundation by the Government of Curaçao.F. fragum larvae settled on either tetrapod design. This resulted in 60 Type I and 64 Type II SUs that, immediately after larvae settled, harboured from 5 to 48 and 8 to 63 settlers with an average of 21.2 (±1.2SE) and 28.0 (±1.7SE) settlers, respectively. F. fragum larvae settled in slightly higher densities (number of settlers per cm2) on Type I than on Type II tetrapods . Larvae settled foremost on the undersides of tetrapod Type II , but did not discriminate between surface orientations on Type I tetrapods . For both tetrapod types, settlement rates inside grooves were 2.4 (Type I) and 2.9 (Type II) times higher than on flat surfaces .An average of 70%  of 1 = 8.6, p = 0.006, Supplementary Table Tetrapods dispersed most during the first two weeks after outplanting with an average of 6.0 cm per week , after which they moved less than 2.0 cm per week  in low complexity habitats Table  Fisher’. TetrapoF. fragum settlers (n = 30 substrates) had survived and grown to an average size of 30.2 mm2 . At that point, 62% of live individuals had completed at least one polyp division and consisted of 2 to 7 polyps. The average settler survival . Growth was also equal between the two designs , suggesting that the grooves served as sheltered microhabitats for newly settled corals. While survival rates of coral settlers on tetrapod Type I were unaffected by the distance that SUs had moved during the study period, 21.3% and 26.6% of the variation in settler survival rates on Type II tetrapods could be linked to the latter’s total dispersal after respectively 3 and 6 months . In this study we examined the possibility to improve the cost-effectiveness of outplanting sexually propagated corals by reducing the labour required to manually outplant them on the reef. We tested two tetrapod-shaped substrates for coral settlement, tetrapod Type I and II  during the first two weeks after outplanting, they rapidly became stuck thereafter 22, allowing the application of coral restoration across much larger scales.Overall, the new tetrapod-shaped substrates could be outplanted efficiently with low costs for labour and materials, enabling 10,000 SUs to be seeded in one hectare of reef within 48 h at a cost of $7,000 USD Table . This reF. fragum settlers was only 9.6% after one year , and very low compared to the 42% survival reported for F. fragum settlers settled on CCA chips in Belize30. While the tetrapods were successfully colonized by thin CCA communities that facilitate larval settlement and metamorphosis, their light-exposed upper surfaces became rapidly overgrown by algal turfs once outplanted on the reef  from non-porous materials such as glass or glazed ceramics, rather than from concrete, could prevent the formation of turf algal communities on the tetrapods, and subsequently enhance the survival and growth of settled corals.While the tested tetrapod designs reduced the amount of labour and costs during the outplanting phase, they were not optimal for coral settler survival and growth. For example, the average survival of Favia fragum larvae preferentially settled inside the tetrapods’ grooves where they experienced lower post-settlement mortality rates. Grooves provide spatial refuges from incidental grazing of newly settled corals by herbivorous fishes and urchins28, and should therefore always be considered in settlement substrate designs to enhance settler survival8. Grooved surfaces accounted for less than a third of the current tetrapods’ total surface area , rather than fully cryptic surfaces such as the undersides of the tested tetrapods, would likely represent better microhabitats to be included in future tetrapod designs to allow a certain degree of protection to new settlers, without compromising their chances to grow into light-exposed areas.Because coral individuals that remain as single polyps past the age of one year often no longer enter the two or more polyp stage, the survival of one-polyp settlers per se is not indicative of effective recruitment4, and as a result has only been applied on small scales . By avoiding the need for outplanting corals using binding materials, the seeding approach allows the deployment of large numbers of young corals in a very short amount of time and at low cost. This technique was most effective in reefs with moderate to high topographic complexity, where tetrapods rapidly became stabilized within the reef framework and resulted in a high SU yield relative to traditional outplanting methods. While we acknowledge that improvements can still be made in future tetrapod designs to optimize the survival and growth of coral settlers, this novel approach nonetheless represents a next step towards large-scale restoration using sexually propagated corals.Sexually propagating corals to restore depauperate coral populations has thus far been a time consuming, technically challenging and an expensive undertakingSupplementary figuresSupplementary tablesSupplementary dataset


The affordable and reliable detection of Hepatitis C Virus (HCV) RNA is a cornerstone in the management and control of infection, affecting approximately 3% of the global population. However, the existing technologies are expensive, labor intensive and time consuming, posing significant limitations to their wide-scale exploitation, particularly in economically deprived populations. Here, we utilized the unique optical and physicochemical properties of gold nanoparticles (AuNPs) to develop a novel assay platform shown to be rapid and robust in sensing and quantifying unamplified HCV RNA in clinical samples. The assay is based on inducing aggregation of citrate AuNPs decorated with a specific nucleic acid probe. Two types of cationic AuNPs, cysteamine and CTAB capped, were compared to achieve maximum assay performance. The technology is simple, rapid, cost effective and quantitative with 93.3% sensitivity, high specificity and detection limit of 4.57 IU/µl. Finally, our data suggest that RNA folding impact the aggregation behavior of the functionalized AuNPs, with broader applications in other nucleic acid detection technologies.  •Direct detection of unamplified HCV RNA in clinical samples.•The Biosensor platform is dependent on the RNA folding behavior.•The developed platform is qualitative, quantitative, and independent on the pH and/or the source of the sample.•The biosensor platform could be easily automated.•First time to use gold nanoparticles in inducing aggregation of gold nanoparticles for unamplified nucleic acids detection and quantification. It was HCV diagnosis is achieved by serologic and Nucleic Acid Testings (NATs) . A majorThe unique optical properties of gold nanoparticles (AuNPs), were originated from their strong Surface Plasmon Resonance (SPR) phenomena, which is responsible for their intense colors, and high extinction coefficient compared to conventional dyes . Thus, AThe non-cross linking method was first introduced by Sato and co-workers , which iHerein, we utilized positively charged gold nanoparticles (cysteamine and CTAB AuNPs) to induce aggregation of citrate capped AuNPs decorated with an HCV RNA specific probe  ˃99%, Dithiothrietol, Dibasic and mono basic phosphate, Sodium dodecyl sulfate (SDS), Sodium Chloride, Sodium borohydride, Hexadecyltrimethyl ammonium bromide (CTAB), Tri-sodium citrate dehydrate, and Ribonuclease A were purchased from Sigma- Aldrich. Cysteamine (2-Mercaptoethylemine HCl ˃98% was purchased from Acros Organics). SV-Total RNA isolation system was purchased from Promega. Artus HCV RG RT-PCR Kit and QIAmp viral RNA kit were purchased from Qiagen. RPMI-1640 medium, L-glutamine, Penicillin/streptomycin was purchased from Lonza, while fetal bovine serum from Gibco. NAP-5 columns .Hydrogen tetracloroaurate (III) trihydrate  was used for size and potential measurements. High resolution transmission electron microscope  was used for imaging2.2Sample Collection was done following ethical approval and informed consent of all subjects. HCV infected serum samples were collected (n=28), which included 23 samples with chronic HCV infection and 5 samples with acute HCV infection. Healthy volunteers provided 17 serum samples. Rapid HCV and Hepatitis B virus (HBV) antibody tests were performed for all samples. Only one sample was HBV positive. Real-time PCR was conducted for all the samples using Artus HCV RG RT-PCR Kit according to the manufacturers’ instructions.2.3Citrate AuNPs was prepared by the traditional sodium citrate reduction method of Gold (III) chloride . Charact2.4Cysteamine AuNPs were synthesized using sodium borohydride reduction method as described . Cysteam2.5CTAB AuNPs synthesis is generally performed following the established seed-mediated growth using two solutions . Herein,2.6HCV RNA extraction was performed using Promega SV-total RNA isolation system according to the modified HCV extraction manufacturer's protocol . The QI-2.72 in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin. Total RNA extraction was conducted using Promega SV-total RNA isolation system following manufacture instructions and each transcript was further purified by homemade magnetic nanoparticles as described previously  were cultured at 37 °C and 5% COeviously , to enha2.8The assay was performed by mixing 5 µl of nanoprobe with 10 µl of RNA sample, heated at 95 °C for 3 min, incubated at room temperature for 5–10 min. Cationic AuNPs (10 µl) was then added to the solution and mixed well. Solution color was developed immediately and observed by naked eye while mixing. The color was stable for ~30 min, depending on the RNA concentration. Solutions were scanned from 400 nm to 750 nm using spectrophotometer. To confirm that the color change is due to RNA solely, the nano-assay was performed on two HCV positive samples before and after digestion with Ribonuclease A enzyme. Each sample was divided into two portions, one portion was treated with the enzyme by adding 10 µl of 10 mg/ml Ribonuclease A to 35 µl of HCV RNA and then incubated for 20 min at room temperature. After completion of the digestion process, the assay was performed on the Ribonuclease A treated sample and on the mock-treated sample. The color of the two samples was then observed and samples were also analyzed using TEM.2.9650 (A530/ A650) was recorded and used to generate the standard curve, in which the A530/ A650 ratio was plotted against the viral RNA log concentration. All HCV RNA samples viral load were determined by the nano-assay using the generated standard curve, from their respective A530/ A650 ratios, using the equation generated by the standard curve.. The results were expressed in IU/µl and converted to IU/ml by )., and examined using the nano-assay. The spectral absorbance for each concentration was scanned spectrophotometrically in duplicate, and the ratio of the non-aggregated nanoparticles at λ530 to the aggregated nanoparticles at λ2.10TEM analysis was conducted immediately after performing the nano-assay for both HCV positive and negative samples. Moreover, it was performed on the Ribonuclease treated and mock-treated sample as described in 33.1max at 520 nm, while that of the nanoprobe showed a slight red shift to 530 nm  cation, which ultimately increases false negative possibilities. Therefore, we decided to use cysteamine-AuNPs that are capable of acting as hydrogen bond donors/acceptors towards the nanoprobe-HCV RNA. This also led to improved color detection since CTAB AuNPs colors were fainter  nanoprobe and cationic AuNPs concentration and type, (2) pH of the reaction mixture, (3) temperature and (4) RNA folding. The nanoprobe density of ~120 probes per small sized AuNPs ~20 nm is known to improve its stability against aggregation and provides acceptable amount for proper probe/RNA hybridization . Final nSolution pH had no impact on the nano-assay as the CTAB AuNPs quaternary ammonium group is always positively charged regardless of the pH while the cysteamine protonated amino group positive charge predominates from pH 9.3, which is way above the assay pH at 7.4. The non-pH dependency of our assay is superior to the non-cross linking assays described previously where pH is a main player for assay performance, especially in the target/mismatched probes . TherefoWe propose RNA folding adaptation to coat the nanoprobe after hybridization, resulting in multiple layers of RNA, thereby stabilizing the nanoprobe. The shape adopted by the re-folded RNA is predicted to give high surface area for the cationic AuNPs to be distributed along the RNA molecules, preventing its interaction with the nanoprobe and preserving their distribution. This notion was confirmed by comparing TEM results for HCV positive and negative samples a and b. 3.6max height at wavelength 530 nm proportionally increased, representing the non-aggregated AuNPs . Thus, at high RNA concentration the ratio of the non-aggregated/aggregated is high, indicating the predominance of the dispersed nanoprobes and their stability and vice versa. The standard curve . All sample concentrations were calculated using the standard curve and ratio described above. Also, some samples with different concentrations were plotted on the standard curve in 2=0.966). All samples quantified by the nano-assay were compared to the gold standard Real-Time RT-PCR method for HCV quantification (As the concentration of the RNA increased the nanoprobe λrd curve b was plofication . The Rea3.7HCV positive samples charge was negative indicating the distribution and spreading of cationic AuNPs along the RNA molecules, thereby preventing the aggregation of the nanoprobe . In agre4We developed a novel AuNPs quantitative biosensor colorimetric assay for the direct detection of unamplified HCV RNA in clinical samples and DNA repair transcripts from cell lines. Stability or aggregations of the nanoprobes rely on cationic AuNPs and RNA target folding rather than the medium ionic strength or pH, which limits false positives. The assay is simple with a turnover time of ~30 min including RNA extraction, sensitive, specific, cost effective and could readily be adopted for full automation.


New vaccine platforms are needed to address the time gap between pathogen emergence and vaccine licensure. RNA-based vaccines are an attractive candidate for this role: they are safe, are produced cell free, and can be rapidly generated in response to pathogen emergence. Two RNA vaccine platforms are available: synthetic mRNA molecules encoding only the antigen of interest and self-amplifying RNA (sa-RNA). sa-RNA is virally derived and encodes both the antigen of interest and proteins enabling RNA vaccine replication. Both platforms have been shown to induce an immune response, but it is not clear which approach is optimal. In the current studies, we compared synthetic mRNA and sa-RNA expressing influenza virus hemagglutinin. Both platforms were protective, but equivalent levels of protection were achieved using 1.25 μg sa-RNA compared to 80 μg mRNA . Having determined that sa-RNA was more effective than mRNA, we tested hemagglutinin from three strains of influenza H1N1, H3N2 (X31), and B (Massachusetts) as sa-RNA vaccines, and all protected against challenge infection. When sa-RNA was combined in a trivalent formulation, it protected against sequential H1N1 and H3N2 challenges. From this we conclude that sa-RNA is a promising platform for vaccines against viral diseases.  RNA vaccines are a promising new approach to control infectious disease. When we compared two influenza RNA vaccine platforms, we observed equivalent levels of protection using less self-amplifying RNA (1.25 μg) compared with synthetic mRNA (80 μg). In addition, a low-dose trivalent self-amplifying RNA protected against sequential H1N1 and H3N2 influenza challenges. Today’s vaccine systems lack important features to be fast or flexible enough to respond to today’s challenges in pathogen control and new systems are urgently needed, especially for epidemics and pandemics. Because of increased globalization and access to rural areas, the emergence of new pathogens is increasingUsing a cellular-like mRNA encoding the protein of interest (synthetic mRNA) there is an immediate translation of the antigen. Though good antiviral protection has been shown,Orthomyxoviridae and can be divided into 3 genera, influenza A, B, and C. Because of their segmented RNA genome, many subtypes exist, especially within the influenza A viruses. Mutation and recombination of different virus subtypes occurs fairly easily leading to the frequent emergence of novel strains. In humans, influenza viruses caused 3 pandemics in the 20th Century. The most recent “swine flu” pandemic in 2009 was considered a low-pathogenicity strain but still infected approximately 200 million people and caused an estimated 201,200 fatalities.One pathogen with pandemic potential is influenza virus, which belongs to the family of In the current study, we looked at the possibility of replacing the protein based seasonal influenza trivalent vaccine with an RNA vaccine. The first question is which RNA vaccine platform was best, synthetic mRNA or sa-RNA. While it is more immunogenic, the production process and stability of the sa-RNA product is more challenging because of the length of constructs. We compared synthetic mRNA and sa-RNA encoding the hemagglutinin (HA) gene from a model influenza virus strain. 64-fold less sa-RNA material was required to induce a similar level of protection, namely 80 μg mRNA versus 0.05 μg sa-RNA was needed for full survival. We then developed and tested sa-RNA encoding HA from seasonal influenza virus A and B strains and observed that they were protective both singly and as a trivalent formulation.To determine the protective potential of synthetic mRNA, BALB/c mice were immunized intramuscularly (i.m.) with a prime-boost regime of 120, 80, or 20 μg synthetic mRNA encoding HA from the H1N1 influenza virus A/Puerto Rico/8/1934 (H1N1/PR8), inactivated virus was used as a positive control. Antibody responses were assessed by hemagglutination inhibition (HAI) A or virain situ prior to inducing an immune response. We investigated whether differences in expression could explain the difference in the dose required for mRNA or sa-RNA immunization. Upon i.m. application of 4 μg mRNA or sa-RNA encoding firefly luciferase, we observed substantial differences between the two RNA vaccine types . Next, we analyzed whether this formulation improved sa-RNA vaccination. BALB/c mice were immunized at days 0 and 21 with 1.25 μg sa-RNA encoding the HA of H1N1/PR8 either formulated with PEI or non-formulated . A third group received only ringer-lactate as a buffer control. All animals immunized with sa-RNA developed an immune response against the HA analyzed by VNT. Both formulated and non-formulated RNA induced VNT response at days 19 -based delivery vehicles. days 19 A after t days 19 B. Taken th of the human dose of a licensed protein-based seasonal influenza vaccine  containing 0.6 μg HA of each A/Califonia/07/2009 , A/Texas/50/2012 (Tx50 H3N2), B/Massachusetts/2/2012 (B/Mass). As a negative control another group received 1.5 μg HIV gp140 sa-RNA to exclude unspecific sa-RNA effects leading to an HA-specific immune response. Mice were immunized with either 1.5 or 0.5 μg PEI-formulated sa-RNA encoding Cal’09 H1N1-HA. Serum samples were taken at weeks 3 (immediately prior to boost), 5, and 7 after the priming immunization. Total anti-H1N1 immunoglobulin G (IgG) was determined by ELISA  Cal’09 H1N1 4 weeks after the boost. sa-RNA-immunized mice were protected against influenza induced weight loss, with significantly less weight loss from day 3 post-infection compared to the control immunized mice (p < 0.05) (+ T cells in the lungs on d7 after infection than either the protein vaccine or the control group (p < 0.01)  B. Influe < 0.05) C, demons < 0.05) D. Vaccin < 0.05) E, sugges < 0.01) F. Taken To check whether the regime described above could be extended to other strains of influenza, the prime-boost study was repeated using a sa-RNA vaccine encoding HA from B/Massachusetts/2/2012 formulated in PEI. The protein vaccine induced significantly higher IgG levels than the HIV RNA control at week 5, furthering increasing by week 7 G. Very lThe current seasonal H3N2 is human adapted and only able to bind α2,6-linked sialic acidThe current influenza vaccine licensed for use—the trivalent protein vaccine used in this study—contains HA from three viral strains. Thus, we wished to test an equivalent trivalent RNA vaccine, to determine whether combining RNA expressed antigens altered immunogenicity. The prime-boost regime used above was adapted such that mice either received 1.5 μg Cal’09 H1N1 HA PEI formulated sa-RNA alone, or a trivalent PEI formulated sa-RNA vaccine containing 1.5 μg each of RNA encoding HA from A/Califonia/07/2009 (H1N1), A/Hong Kong/1/68  and B/Massachusetts/2/2012. Control mice were unvaccinated (naive) or received protein vaccine. Antibody responses against the encoded antigens were measured in sera on day 7 after infection A–5C. Tri5 copies/μg in naive mice (p < 0.001)  B. Prior < 0.001) C. The sa< 0.001) D. Intere< 0.001) E. Thus, In this study we have demonstrated that sa-RNA vaccines protect against influenza A or B infection when administered singly or in trivalent combination. Compared to non-amplifying synthetic mRNA vaccines, sa-RNA vaccines induced protection with a 64-fold lower dose, which may result from prolonged and increased transgene expression. Previous studies have demonstrated the efficacy of high-dose mRNA vaccinesIn-vitro-transcribed RNA is recognized by the host cells by a number of pattern recognition receptors, RNA-dependent protein kinase (PKR),We show here that 64-fold less sa-RNA is required to achieve the same level of protection than non-amplifying synthetic -mRNA. On a per-gene basis the dose delivered is even smaller for the sa-RNA group as the sa-RNA construct is larger than the mRNA, 9,300 compared to 2,200 nt. The difference in dose required for protection is important for translation into clinical practice as a significant scale up of the total dose is needed for equivalence in human studies.One of the aims of the study was to determine whether combining antigens in an RNA vaccine would affect the efficacy of the vaccine. There were two possible causes of interference: inflammation and antigenic sin. It was possible that delivering more RNA would increase inflammation and shut down cellular translation machinery via type I interferon (IFN) and PKR.The sa-RNA vaccines were also compared to DNA, delivered with electroporation or formulated with PEI. It was of interest because while it induced lower antibody responses, formulated DNA without electroporation was still protective. This protection may have resulted from the H1-specific T cell induction, which was significantly greater after the DNA vaccination; we have shown that DNA vaccine induced CD8 T cells are protective,in vitro transcription is based on protocols provided by the MEGAscript T7 Transcription Kit . The general procedure starting with linear DNA template containing the T7 promoter, and particularly with respect to co-transcriptional capping with the synthetic cap analog beta-S-ARCA(D1) (used in 4:1 ratio regarding guanosine triphosphate [GTP]), is carried out similarly to as described before.T7 tibialis posterior. Following intraperitoneal (i.p.) injection of 100 mg/kg body weight D-luciferin , inhalation anesthesia (isoflurane 2.5%) was introduced, and, during maintenance, serial images of the animals were taken at the indicated time points using an IVI Spectrum imaging system (PerkinElmer). Photons emitted were collected for 1 min. Bioluminescence intensity from the muscle region of interest was quantified using Living Image software (PerkinElmer).Mice were anesthetized by inhalation anesthesia (isoflurane 2.5%) . Subsequently, 20 μL of pre-mixed RNAs in RNase-free PBS  was injected i.m. to the 50 of infectious influenza virus. The final serum dilution of this assay was 1:1,280 and thereby also the upper detection limit. The serum-virus mix was then applied to confluent Madin-Darby canine kidney (MDCK) monolayer in 96-well plates and incubated for another 3 days. 50 μL of supernatant was thereafter incubated with 50 μL of 0.5% chicken red blood cells , and red blood cell agglutination was evaluated. The VNT titer was recorded as the inverse of the lowest dilution that inhibited agglutination (VNT/50 μL).To determine the level of neutralizing antibodies against HA in the serum of animals, VNTs were performed in accordance with the Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza . A serial dilution of serum samples starting with 1:10 was incubated for 2 hr with 100 TCIDTo determine the serum level of anti-HA antibodies that inhibit hemagglutination in mice, sera were collected and HAI assay was performed following the Manual for the laboratory diagnosis and virological surveillance of influenza . Briefly, serum samples were treated overnight with receptor destroying enzyme II “Seiken” in a 1:5 ratio  followed by heat inactivation for 30 min at 56°C. Sera were used in duplicates and serial dilutions (1:2) were performed before adding 4 hemagglutinating units (HAUs) of H1N1/PR8 virus. After 60 min incubation at room temperature, 50 μL of 0.5% red blood cells (Lohmann Tierzucht) were added and the mixture incubated for 30 min at room temperature before evaluation of agglutination. The HAI titer was recorded as the inverse of the lowest dilution that inhibited agglutination (HAI/50 μL).anterior tibialis with 20 μL Begripal 2014/15 (Novartis Vaccines) containing 0.6 μg HA from each of B/Massachusetts/2/2012, A/Texas/50/2012 (H3N2)-like, and A/California/7/2009 (H1N1) pdm09-like viruses. For synthetic RNA vaccination, mice were injected i.m. with 20 μL non-formulated mRNA, non-formulated, or formulated sa-RNA. For DNA vaccination, mice received 1.5 μg formulated DNA or naked DNA with electroporation i.m. Where used, two lots of 5 pulses of 150 V with switched polarity between pulses were delivered using a CUY21 EDIT system . For infections, mice were either anesthetized with ketamin-rompun before infected intranasally (i.n.) with 30 μL containing 2.4 × 104 PFU (10x MLD50) of H1N1/PR8 or anesthetized using isoflurane followed by i.n. application with 3 × 104 PFU A/California/7/2009 (H1N1) influenza.6- to 8-week-old female BALB/c mice were obtained from Harlan UK  or Janvier  and kept in specific pathogen-free (SPF) conditions in accordance with the German animal welfare law and United Kingdom’s Home Office guidelines. All work was approved by the Animal Welfare and Ethical Review Board (AWERB) at Imperial College London or by the local animal welfare committee of Rhineland-Palatinate (reference number G-13-8-063). For the inactivated virus control group, mice were immunized with 10 μg per 20 μL H1N1/PR8 virus , and for licensed protein vaccine groups, mice were immunized i.m. via the H1N1 influenza virus A/Puerto Rico/9/1934 was a kind gift from Veronika von Messling  and thereafter grown in-house on MDCK cells in fetal calf serum (FCS)-free minimum essential media (MEM) . H1N1 influenza (strain A/England/195/2009), isolated by Public Health England in the United Kingdom, April 2009,2SO4 as stop solution was used to detect the response, and optical densities were read at 450 nm.Antibodies specific to influenza H1N1 were measured in sera using a standardized ELISA. MaxiSorp 96-well plates (Nunc) were coated with 1 μg/mL H1N1 surface protein or a combination of anti-murine lambda and kappa-light-chain-specific antibodies  and incubated overnight at 4°C. Plates were blocked with 1% BSA in PBS. Bound IgG was detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (AbD Serotec). Alternatively, IgG1 or IgG2a were detected using subtype-specific secondary antibodies. A dilution series of recombinant murine immunoglobulin was used as a standard to quantify specific antibodies. 3,3',5,5'-Tetramethylbenzidine (TMB) with Hg for 5 min. Supernatants were removed, and the cell pellet treated with red blood cell lysis buffer  before centrifugation at 200 × g for 5 min. The remaining cells were resuspended in RPMI 1640 medium with 10% fetal calf serum, and viable cell numbers were determined by trypan blue exclusion.At specified time points post-immunization, blood samples were taken by tail-vein bleed, and sera were isolated after clotting by centrifugation. Mice were culled using 100 μL intraperitoneal pentobarbitone , and tissues were collected as previously described.in vivo was assessed by Trizol extraction of RNA from frozen lung tissue disrupted in a TissueLyzer . RNA was converted into cDNA, and qRT-PCR was carried out using bulk viral RNA for the influenza M gene and mRNA using a 0.1-μM forward primer (5′-AAGACAAGACCAATYCTGTCACCTCT-3′), a 0.1-μM reverse primer (5′-TCTACGYTGCAGTCCYCGCT-3′), and a 0.2-μM probe (5′-FAM-TYACGCTCACCGTGCCCAGTG-TAMRA-3′) on a Stratagene Mx3005p . M-specific RNA copy number was determined using an influenza M gene standard plasmid.Viral load 533-541 IYSTVASSL Pentamer R-PE , CD3-FITC , CD4- PE/Cy7 , and CD8-APC-H7 (BD). Analysis was performed on an LSRFortessa Flow Cytometer (BD). Fluorescence minus one (FMO) controls were used for surface stains.Cells were stained with Fixable Violet Dead Cell Stain , washed, suspended in Fc block  in PBS-1% BSA, and then stained with the following surface antibodies: influenza A H1 HACalculations described in the figure legends were performed using Prism (v.6) .L.L., E.K., A.B.V., S.E., D.B., and K.C.R. performed the studies; T.B., M.P., H.H., and L.W. developed and provided the RNA vaccine constructs; A.B.V., U.S., and J.S.T. wrote the paper; and J.S.T., S.T.R., and A.B.V. designed the studies.A.B.V., S.E., H.H., K.C.R., L.W., M.P., T.B., S.T.R., and U.S. are or were employees of Biontech or related companies. All other authors declare no competing financial interests.


Transporter proteins mediate the translocation of substances across the membranes of living cells. Many transport processes are energetically expensive and the cells use 20 to 60% of their energy to power the transportomes. We hypothesized that there may be an evolutionary selection pressure for lower energy transporters.Neorickettsia risticii and Neorickettsia sennetsu, as likely origins of the mitochondrion in eukaryotes, based on the phylogenetically restricted presence therein of clear homologues of modern mitochondrial solute carriers.We performed a genome-wide analysis of the compositional reshaping of the transportomes across the kingdoms of bacteria, archaea, and eukarya. We found that the share of ABC transporters is much higher in bacteria and archaea (ca. 27% of the transportome) than in primitive eukaryotes (13%), algae and plants (10%) and in fungi and animals (5–6%). This decrease is compensated by an increased occurrence of secondary transporters and ion channels. The share of ion channels is particularly high in animals (ca. 30% of the transportome) and algae and plants with (ca. 13%), when compared to bacteria and archaea with only 6–7%. Therefore, our results show a move to a preference for the low-energy-demanding transporters (ion channels and carriers) over the more energy-costly transporter classes  as part of the transition from prokaryotes to eukaryotes. The transportome analysis also indicated seven bacterial species, including The results indicate that the transportomes of eukaryotes evolved strongly towards a higher energetic efficiency, as ATP-dependent transporters diminished and secondary transporters and ion channels proliferated. These changes have likely been important in the development of tissues performing energetically costly cellular functions.The online version of this article (10.1186/s12864-018-4816-5) contains supplementary material, which is available to authorized users. The expansion of life on Earth has involved competition and also cooperation among organisms for the utilisation of resources which have been accessible to them , 2. Argu, ion channels, secondary transporters, and ATP-dependent transporters. To translocate substrates, ATP-dependent transporters bind and hydrolyse ATP [Despite the importance of cellular transportomes , transplyse ATP , ion chalyse ATP .To study the compositional changes of transportomes, we analysed the transportomes of 249 evolutionarily distant species (of which 222 were annotated in this study) from archaea, bacteria and eukarya. These included 126 prokaryotic species (from 16 taxonomic phyla and 60 taxonomic orders), 30 primitive eukaryotes , 30 algal and plant species, 30 fungal species, and 33 animal species  = 0.937) with the exception of primitive eukarya, where the transportome-encoding proportion of genes was the lowest  as the average required energy per single substrate translocation. We calculated the EUE values for the transportomes studied (more details in the Methods section). The EUE describes the overall energetic performance of transportomes at organismal level and most importantly it does not indicate the total energy consumption by the cellular transportome, because the latter depends also on the flux through individual transporters that is largely unknown. In contrast to the total energy requirements, the EUE is therefore not subjected to spatiotemporal variations. By comparing the average EUEs of the transportomes across the different domains of life, we found that the EUE has improved in eukaryotes by reductions of up to 0.50 ATP in the average ATP consumption per single transport event mediated by transporters Table .Table 1IFurthermore, the data suggest that animals have mainly relied on the diversification of ion channels, fungi on secondary transporters, and finally, algae and plants on both transporter classes for the evolution of their transportomes Fig. . For enehttp://www.membranetransport.org/transportDB2/index.html). This extended our prokaryotes to 2637 transportomes :proton antiporter-3 family, the branched chain amino acid:cation symporter family, the NhaB Na+:H+ antiporter family, the riboflavin transporter family, and the Na+-dependent bicarbonate transporter family  as the most likely origin of the mitochondrion in eukaryotes [The analyses also indicate family member expansions for secondary transporters  and ion channels . To find prokaryotic- and eukaryotic-specific families, we further enriched our analysis by including the publicly-available data for the secondary transporters and ion channels of prokaryotes found at TransportDB 2.0  coding genes. Since other evidence had also suggested Rickettsiales as candidates for this, our transoportome findings strengthen considerably the case for such a lineage.The present inter-kingdom comparison of transportomes provides genome-scale molecular evidence for their evolution towards an improved energetic efficiency. This has likely been very influential due to the high energy demand of the cellular transport machinery and also for the development of tissues performing energetically costly functions. The present data also strengthen members of the previously reported bacterial order Rickettsiales as the origin of the mitochondrion by recognising http://www.membranetransport.org/transportDB2/) [The publicly-available membrane transporter data on ion channels and secondary transporters were extracted from TransportDB ortDB2/)  . The tr/  . The ortDB2/) . We alsoortDB2/) , and thehttp://www.ensembl.org/index.html, http://fungi.ensembl.org/index.html, http://protists.ensembl.org/index.html, http://plants.ensembl.org/index.html) [To predict the transportomes, the proteomes of organisms were downloaded from the Genbank and Ensembl databases (ex.html) . All proTo make an approximation of the energetic performance of the cellular transportomes, we calculated the inter-transportome changes in energy-usage efficiency (EUE), which we defined as the average free energy demand for a single transport event. The following assumptions were made when calculating EUE values. Equilibrative ion channels require no free energy for the transport action. While some of the secondary transporters also act in an energy-independent manner known as equilibrative transport or facilitated diffusion, others exploit the electrochemical potential established by ATPases across membranes , 68–70. Additional file 1:Table S1. The list of organisms with publicly-available data on the ion channels and secondary transporters. (DOCX 168 kb)Additional file 2:Data S1. Genomic and transportome data on the organisms included in the study. (XLSX 255 kb)


Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils. Tau is a common neuronal protein that, under circumstances and conditions not well understood to date, self-assembles into intracellular aggregates in several neurodegenerative diseases including Alzheimer disease. These aggregates are formed of fibrous polymers. The mechanism by which this critical transition from a soluble protein to insoluble fibrous material occurs is unknown. We have discovered a novel state in which many tau molecules become compacted into a protein-rich droplet while maintaining their solubility and native-like protein conformations. Chemists refer to this dense liquid droplet state as a complex coacervate phase, and it is held together by the opposite charges of their constituents, ions, and water. In the case of the tau protein, the oppositely charged constituent is RNA. Indeed, we found that in human neuronal cell culture, tau selectively binds to a category of RNA known as tRNA. Interestingly, tau and RNA favorably condense to a complex coacervate phase when the charges between them are matched and at elevated temperatures, such that tau-RNA droplets could be observed at physiologically viable protein concentrations simply by increasing the temperature from room to physiological temperatures. When the tau-RNA–dense droplets are incubated together over time, tau transitions to a conformation similar to that found in pathological fibers. Our experiments therefore demonstrate physicochemical properties of tau that may predispose it to undergo changes associated with neurodegenerative disease. Inclusions consisting of the tau protein occur in many neurological conditions with Alzheimer disease the most prominent among them. Normally, tau is in a dynamic equilibrium between a microtubule-bound and free state. Under disease conditions tau self-assembles into fibrils that eventually lead to highly insoluble polymeric inclusions known as neurofibrillary tangles. The underlying biophysical basis for the transition of tau from a microtubule-associated protein to an insoluble fibril is unknown. However, a clue comes from the observation that polyanions, such as heparin, promote tau fibrillization . AlthougOur experiments began with the finding that tau can bind RNA in living cells. Interestingly tau-RNA binding showed selectivity for tRNAs. This observation along with the known categorization of tau as intrinsically disordered and its ability to spread from cell to cell in a manner that resembles prions , 5 suggeHere, we show that tau-RNA complexation can lead to complex coacervation. When multiple tau molecules weakly bind RNA, and overall charge matching is achieved between the polycation, tau, and polyanion, RNA, tau undergoes reversible condensation and LLPS into micrometer-sized droplets. Remarkably, within this LLPS state, tau maintains a high internal segment mobility and a locally compact conformation that protects the core region of tau known as PHF6(*), as found in dilute solution state, despite the molecular crowding associated with coacervation. In contrast, this region experiences full extension that exposes the PHF6(*) region to the solvent and stacks into β-sheets in the presence of a different polyanion, heparin, that induces irreversible fibrillization of tau . The spoPhotoActivatable Ribonucleoside-enhanced Individual-nucleotide resolution UV Cross-Linking ImmunoPrecipitation and high-throughput sequencing (PAR-iCLIP) using the human tau-specific antibody, HJ 8.5. The tau constructs and tau mutants used in this study are shown in 32P-labeled RNA bands correspond to immunoprecipitated cross-linked tau-RNA complexes  studies, particularly for atypical RNA binding proteins [To identify the types of RNA crosslinked to tau, DNA libraries were prepared from the immunoprecipitated radiolabeled bands and sequenced. We analyzed the distribution of tau-bound RNA from the human genome by defining 8 regions: exons, introns, long intergenic noncoding RNAs (lincRNAs), small nuclear RNAs (snRNAs), rRNAs, microRNAs (miRNAs), tRNAs, and intergenics. In tau-expressing HEK cells, tRNAs were overwhelmingly the highest category of RNA crosslinked to tau . Endogenproteins . Despiteproteins , 32–35. Arg  for 4R2N tau binding to tRNA of 460 ± 47 nM  induced a shift in unacetylated tRNALys , yieldin ± 47 nM . The derectively . The dis2R) complexes, while at higher tau:tRNA molar ratios, tau and tRNA form larger protein multimer-RNA (P2nR) complexes with 2n reaching as high as 6. This implies that tRNA is capable of binding multiple tau proteins in a multistep process. Interestingly, when the tau:tRNA ratio was decreased by increasing the tRNA concentration relative to tau, the higher order P2nR complexes dissociated to P2R, maintaining a tau dimer bound to RNA in the presence of excess (7.5-fold) RNA  complex forms first followed by the formation of a protein multimer-RNA (P2nR) complex, is in fact consistent with the model for RNA binding to the protein AUF1 [The gel shift assay showed multiple bands corresponding to different tau:tRNA stoichiometries corresponding to high molecular weight protein-RNA complexes . The fraold) RNA . Such hiein AUF1  and hnRNein AUF1 .Mixing of 4R2N or Δtau187  RNA molar ratios from 33:1 to 330:1 and Δtau187:poly(U) RNA ratios from 267:1 to 333:1  regions offer a powerful tool to compare the conformational state of tau in dilute solution, droplet and aggregation-induced states. We prepared a Δtau187 G272C/S285C-SL2 construct that was doubly MTSL-labeled at sites 272 and 285 (see * hexapeptide region by double electron-electron resonance (DEER) spectroscopy. Surprisingly, the mean distance flanking the PHF6* region remained unchanged from dilute solution state to when tau was condensed into a concentrated complex coacervate phase in association with poly(A) RNA and tRNA (compare blue versus red trace in (*) segment adopts when neatly stacked in β-sheets  fluorescence under tau-poly(U) RNA droplet forming conditions that gradually increased over 15 hours (see Δtau187 + poly(U)). The ThT fluorescence, however, was nearly eliminated at increased salt concentration that corresponded to conditions under which tau-RNA associations are weakened and droplets are dispersed. However, even after 15 hours of incubation, the ThT fluorescence intensity from the tau-RNA droplet samples was significantly less (less than 5%) compared to what was observed in the presence of the aggregation-inducing heparin, under similar charge ratio and mass concentration and following a brief incubation time of less than 20 minutes , 4R2N tau harboring the P301L mutation (1sample), and 4R1N tau harboring the P301L mutation fused to CFP (2 samples). Four samples were neurons derived from hiPSCs obtained by reprogramming dermal fibroblasts with virally transduced Yamanaka factors [tau, 2 harboring a P301L tau mutation, and 1 harboring the A152T variant [6 per 10 cm dish, and the cells switched to neurobasal medium the next day and differentiated for 7 days using 10 μM retinoic acid.A total of 8 samples were used for PAR-iCLIP studies. Four samples were HEK (HEK 293T) cells that expressed either 4R2N (residues 1–441) wild-type  factors  that exp variant , 60. The variant . When ne1 Alexa 680, ; mouse IgG Alexa 680 ; rabbit IgG 800 .The tau antibodies used were Tau-5 for probing total tau in western blot ; human tau-specific HJ 8.5 and mouse tau-specific HJ 9.2  for CLIP; PHF-1  for sarkosyl-insoluble tau. Other antibodies used are MAP2 antibody ; CDK5 rabbit antibody ; GFP rabbit antibody ; β-Actin ; mouse IgG2 of 365 nm UV light on ice. 4-SU can enhance the cross-linking efficiency especially for proteins in the cytoplasm [32P-labeled RNA band was run on a polyacrylamide Tris-Borate Urea (TBU) denaturing gel to demonstrate and confirm the size of the complex.PAR-iCLIP was applied to detect specific RNAs bound to tau in living cells , 32. Celytoplasm , 30, 32.ytoplasm  raised ahttps://github.com/marcelm/cutadapt/) [http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) [tRNAscan-SE v.1.23. The secondary structure for each tRNA was obtained from GtRNAdb (http://gtrnadb.ucsc.edu/).The iCLIP libraries contained an experimental and a random barcode, which allowed multiplexing and the removal of PCR duplicates. After the barcodes were introduced, sample and control from 1 set of experiments were mixed to remove batch-to-batch variation. Libraries were sequenced on an Ion Torrent Proton . Fastx collapser from FASTX-Toolkit was used to collapse reads and filter replicates resulting from the PCR based on the random barcode. Reads were separated into samples by the barcodes at the 5ʹ ends of reads. The reverse transcription primers sequenced at both ends of the reads were trimmed with Cutadapt 1.9 (tadapt/) . After tx.shtml) . RseQC [x.shtml)  was usedx.shtml)  were usex.shtml) . IndividSmall RNAs were extracted from HEK cells and hiPSC-derived neurons using miRNA isolation kit . Library preparation was adapted from the Ion RNA-seq v2  protocol. cDNAs with size range from 30–100 bp were selected and sequenced. Reads were aligned to both human genomes and tRNA sequences. When mapping reads to tRNA sequences, the Bowtie read aligner  protocolFull-length recombinant human 4R2N tau, N-terminal truncated, microtubule binding domain containing, K18 tau (residues 244–372), and Δtau187 (residues 255–441 with a His-tag at the N-terminus) were used for in vitro studies. Methods for expression and purification of all recombinant tau variants are detailed in Lys (tRNA Probes) in 100 mM sodium acetate buffer at pH 7.0. The molar concentration of tRNALys was accurately remeasured with UV spectrophotometry after base-hydrolization to account for the hyperchromic effect from the secondary and tertiary structure of tRNA [-1cm-1. For the gel shift assay, protein was incubated with tRNA at 37°C for 10 minutes in the presence of 0.5 mM EDTA, 0.5 mM MgCl2, 2 U SUPERase• In RNase Inhibitor , 0.01% IGEPAL CA-630 (Sigma-Aldrich), and then applied to a TBE 8% Polyacrylamide Gel . After gel separation, tRNA was stained with SYBR Gold II . For quantitative analysis, the fraction of free and bound tRNA was quantified in ImageJ2  [The gel shift assay was performed with recombinant full length 4R2N and K18 tau and chromatographically purified unacetylated yeast tRNA of tRNA . RNA43 wsda, MD) .Full-length tau or K18 tau were dialyzed overnight into a specified buffer for ITC . tRNA  was resuspended in the ITC buffer and concentration determined by using a Nanodrop 1000 . Experiments were run on a Nano ITC , in which 300 μM tRNA was titrated (5 μl injections) into a 1 ml protein solution of 30 μM tau. Data were analyzed by using the NanoAnalyze v3.6 software . After subtracting the heat generated by tRNA titration into an empty buffer, the experimental data were fitted well with an independent binding model, but not with a cooperative nor an independent 2-site model. Fitting to a cooperative or to an independent 2-site model gave a statistical error over 100% for the first Ka value. This could be due to the low enthalpy governing the binding event that is observed in the ITC data, and could possibly be resolved with increased concentrations of the reactants; here, tau and RNA. However, solubility limitations likely preclude these experiments from being realistically achievable. Of note, the second Ka value produced by the 2-site model is very similar to the values obtained from an independent model, suggesting that the predominant interaction is indeed the one we have described. Experiments were repeated in triplicates and standard error of the mean reported.-1mM-1, calculated from extinction coefficient of Tyrosine [2, respectively. In order to achieve spin dilution, Δtau187/322C and Δtau187G272C/S285C were also labeled with the diamagnetic analogue of MTSL probes, and are referred to as Δtau187/322C/322C-DL and Δtau187G272C/S285C-DL2. Images shown in To achieve labeling with paramagnetic or diamagnetic probes, the protein was dissolved in 6 M guanidinium hydrochloride and labeled overnight at 4°C using a 20-fold molar excess of the spin label  methanethiosulfonate  or the diamagnetic analog of MTSL  methanethiosulfonate . Excess label was removed using a PD-10 desalting column  equilibrated in a 20 mM ammonium acetate buffer at pH 7.0. The protein was concentrated using a 3 kDa Amicon centrifugal filter . The final protein concentration was determined by UV-Vis absorption at 274 nm by using an extinction coefficient of 2.8 cmTyrosine . The 2 vDroplets were formed in the 20 mM ammonium acetate buffer with NaCl concentration varying between 0 and 100 mM and glycerol concentration from 0% to 50% v/v. Unless explicitly indicated for measurements at various temperatures, all samples were prepared and characterized at room temperature. Solutions of Δtau187 or full length 4R2N tau was mixed with tRNA , poly(A) RNA, or poly(U) RNA  at varying protein, RNA, NaCl, and glycerol concentrations in a 0.6 ml Eppendorf tube. RNAs were weighted out as powder and the mass concentration was calculated. The tau:RNA mass ratio, the total concentration of tau and RNA, as well as the NaCl salt concentration were optimized to maximize droplet formation, while choosing a total biopolymer density to avoid overlapping of droplets in the images to simplify the calculation of the droplet coverage (%). Microscopy images were acquired at 10 minutes after mixing—the droplets form spontaneously and are expected to be in equilibrium with the dilute supernatant. A concentration of 19% v/v for glycerol/water was determined to be an optimal concentration to ensure cryoprotection for DEER measurements carried out at approximately 80 K, while also ensuring maximal droplet formation at room temperature .Immediately after mixing tau in a 0.6 ml Eppendorf tube with RNA under droplet forming conditions (established above) and ensuring thorough mixing, 1 μl of this mixture was applied to a microscope slide that is closed with a cover slide gapped by 2 layers of double-sided sticky tape to generate a liquid sample region with consistent thickness. The microscope slide was kept at room temperature for 10 minutes with the cover slide facing down, during which the particles within the liquid sample region settled down onto the surface of the cover slide. Images were taken with a 12-bit charge coupled device (CCD) camera across the entire sample liquid region near the surface of the cover slide using an inverted compound microscope . Before imaging, Köhler illumination was applied and the focus close to the surface of the cover slide optimized to enhance the contrast between the dark droplets and the bright background.5 Maleimide (Thermo Fisher) at the same 322 site as Δtau187/322C-SL. 50 μM of Δtau187/322C-FL was mixed with 350 μM Δtau187/322C-SL at a 1:7 molar ratio in order to prevent saturation. 800 μg/ml of poly(A) RNA was further added to this tau solution, resulting in droplet formation in the 20 mM ammonium acetate buffer and in the presence of 20 mM NaCl. 10 μl of the mixture was pipetted and put on a microscope slide with a cover slide gapped by double-sided sticky tape. Confocal images were acquired using a spectral confocal microscope .For confocal microscopy, Δtau187/322C was fluorescence-labeled (Δtau187/322C-FL) with Alexa Fluor 488 Chttps://github.com/yanxianUCSB/DropletAnalysis).To quantify the amount of droplet formed under the given experimental condition of interest, images were taken by a 12-bit CCD camera of an inverted compound microscope , and recorded in TIF format. With illumination and focus optimized, droplets settling on the cover slide have lower intensity than their surrounding on the images. An image of the 20 mM ammonium acetate buffer was taken to calculate the average intensity to set as threshold in order to classify different parts of the image into droplets and buffer. For each image, the area of the droplets was divided by the total area of the image, generating a percent droplet coverage value on the cover slide. Droplets with eccentricity above 0.9 or equivalent diameter below 1μm were filtered out in order to reduce false reading. The MATLAB code is made available as supplementary files on the internet  and a dielectric cavity . Samples were prepared by either mixing 200 μM Δtau187/322C-SL with 300 μM unlabeled Δtau187/322C (to generate 40% spin labeled sample) or by using 500 μM Δtau187/322C-SL (100% labeled). Viscogen was added to the sample to achieve either 19% v/v glycerol (for the droplet samples) or 30% v/v sucrose (for the aggregated samples) matching the DEER conditions. tau samples under droplet forming condition were prepared by adding 1.5 mg/ml RNA, and tau samples under aggregation-inducing conditions prepared by adding 125 μM heparin . A sample of 3.5 μl volume was loaded into a quartz capillary  and sealed at 1 end with critoseal and the other with beeswax, and then placed in the dielectric cavity for measurements. Cw EPR spectra were acquired by using 2 mW of microwave power, 0.3 gauss modulation amplitude, 150 gauss sweep width, and 25 scans for signal averaging.2 with 500 μM analog-labeled Δtau187G272C/S285C-DL2 at a 1:10 molar ratio to achieve spin-dilution and avoid artifacts from unwanted inter-protein spin distances. For DEER, tau samples under droplet forming condition were prepared by adding 1.65 mg/ml RNA and ensuring 19% v/v glycerol concentration, and tau samples under aggregation-inducing conditions prepared by adding 137.5 μM heparin and ensuring 30% v/v sucrose concentration. 40 μL samples containing 550 μM concentration of tau were loaded into a quartz capillary (2.4 mm od x 2 mm id) and flash frozen in liquid nitrogen after 20 minutes of incubation at room temperature and the specific conditions listed for the data. DEER measurements were conducted using the dead-time–free 4-pulse DEER sequence at 80 K, by using 22 ns (π/2) and 44 ns (π) observe pulses and a 30 ns (π) pump pulse. The raw DEER data were processed using Gaussian fitting via DeerAnalysis2013  [DEER measurements were performed on a Q-band pulsed EPR spectrometer operating at 32 GHz  equipped with a QT2 resonator (measurements done courtesy of Bruker Biospin). Samples were prepared by mixing 50 μM Δtau187G272C/S285C-SLzerland) .160 μM Δtau187 was mixed with 480 μg/ml poly(U) RNA or 40 μM heparin and incubated at room temperature in the presence of 4 μM ThT for over 15 hours . The fluPhe -fluorescein transfected in primary mouse neuron at 14 days in vitro by using lipofectamine 2000 transfection reagent  following the manufacturer's protocol  comprise the N-terminal projection domain (residues 1–243), the 4-repeat microtubule binding domain (residues 244–372) and the C-terminal region (residues 373–441). The inserts near the N-terminus−N1, N2−and the second repeat−R2−can be alternatively spliced, giving rise to six isoforms. Two hexapeptide motifs−PHF6* and PHF6−at the beginning of R2 and R3 repeats are known to promote paired helical filament (PHF) aggregation. Cells expressing 4R2N wild type, or mutant (A152T or P301L), or 4R1N P301L fused to CFP were used in PAR-iCLIP study, with the mutation sites marked here with asterisks. Full length 4R2N tau, K18 tau (residues 244–372) and Δtau187 (residues 255–441 with a His-tag at the N-terminus) were used for (TIFF)Click here for additional data file.S2 Fig32P-labelled RNA crosslinked to tau protein in HEK cells expressing tau (a-c) and in neuroblastoma with endogenous tau (d). The experimental conditions are indicated at the top of each lane: CLIP experiments with expressed P301L tau and immunoprecipitated (IP’ed) with human specific tau antibody (HJ 8.5 gift from D. Holtzman) , CLIP experiments with expressed GFP and GFP IP . CLIP experiments with expressed wild type (WT) tau and IP’ed with HJ 8.5 , control without UV crosslinking and IP’ed with HJ 8.5 , control in cells that do not express tau and IP’ed with HJ 8.5 . RNAse as indicated (panel b). CLIP experiments with expressed P301L tau fused to CFP and IP’ed with HJ 8.5 . Controls without UV and without expressed tau . RNAse as indicated did not change the migration of the tau RNA complex (panel c). CLIP experiments with antibody HJ9.2 (gift from D. Holtzman) against mouse tau (mTau), HJ8.5 antibody against human tau, MAP2 antibody and CDK5 antibody . To the left of each phosphor image are the western blots that utilize the same antibodies used for the CLIP IP: GFP and tau (panel a), tau (panel b and c), tau MAP2 and CDK5 (panel d). Below each phosphor image shown are the loading control western blots, where the lysates input for immunoprecipitation are probed by antibody Tau-5 and anti-actin. The RNA-protein complexes marked within the rectangles of panel a were cut from the blot for library preparation and sequencing. Note that two regions from the GFP control were cut and one corresponding to the MW of tau protein and other corresponding to the MW of GFP. (e) PAR-iCLIP-mapped reads from four samples of HEK cells harboring WT, P301L and two P301L-CFP tau. (f) PAR-iCLIP-mapped reads from four samples of hiPSC-derived neurons harboring WT, two P301L and one A152T tau. Data from the higher and lower MW band of the GFP or CDK5 controls are labeled as GFP-H, GFP-L, CDK5-H and CDK5-L, respectively. The non-cross sample is from the control without UV crosslinking. The numerical data used in e-f are included in Phosphor images in the blue frame (a-d) show (TIFF)Click here for additional data file.S3 Fig(a-b) tRNA abundance in CLIP samples vs total small RNA controls in HEK cells (a) and hiPSC-derived neurons (b) indicate that tRNA distributions differ between the total tRNA pool and the CLIP tRNA pool. Specifically, the population above the diagonal is much greater, indicating that the tRNA abundance from PAR-iCLIP samples from tau-bound RNA is significantly greater compared to the total small RNA present in HEK or hiPSC neuron cells. (c) Top 10 ranked tRNAs bound by tau in HEK cell and hiPSC-derived neurons. The numerical data used in a-b are included in (TIFF)Click here for additional data file.S4 FigAccumulative CLIP cDNA reads from all tRNAs recovered in tau PAR-iCLIP of HEK cell and hiPSC-derived neurons (top two graphs) in comparison to the total small RNA population  in HEK cells and hiPSC-derived neurons (bottom two graphs). The anticodon is designated as position 1–3 for alignment purpose, and the tRNA structure is shown below the x-axis in one dimension. Note the distinct pattern of crosslinking sites around the anticodon region in the PAR-iCLIP reads from both the Hek cells and hiPSC neurons in contrast to the expected 5’ bias of the tRNAs from the small RNA-Seq population. The numerical data used are included in (TIFF)Click here for additional data file.S5 Fig(a) Yeast tRNA was titrated into solutions of K18 tau in an ITC experiment. The top panels show the raw incremental-titration data. The area under each peak is integrated and plotted against the tRNA:tau molar ratio and fitted to an independent binding model (the bottom panel). The numerical data are included in (TIFF)Click here for additional data file.S6 Figin vitro. Left: droplet coverage with varying NaCl concentration in samples made of 160 mM Δtau187 and 480 mg/ml poly(U). Right: droplet coverage by varying Δtau187:poly(U) charge ratios with no NaCl added. A total mass concentration of Δtau187 and poly(U) was kept at ~2 mg/ml, with 74 mM Δtau187 and 240 mg/ml poly(U) showing the highest droplet coverage (%). (b) Droplet coverage (%) of Δtau187 and poly(U) RNA with varying glycerol concentration. Experimental condition was maintained with 80 mM Δtau187 and 120 mg/ml poly(U), and with no added NaCl. (c) Calculations of tau-RNA molar or charge ratio, based on their chemical properties (top table), for various experimental conditions where droplet formation is observed (bottom table). The charge of the RNA and protein species at a given pH were estimated  Representative bright-field images of 4R2N tau-RNA droplet at two NaCl concentrations. (e) Left: droplet coverage (%) with varying NaCl concentration, and at a charge ratio of tau 4R2N and RNA at 0.7:1. Right: droplet coverage with varying 4R2N tau:RNA charge ratios, with no NaCl added. A total polymer mass concentration ~3.6 mg/ml was maintained in the experiments in d-e, with 80 mM 4R2N and 349 mg/ml poly(A) showing the highest droplet coverage. All data and images in a-e were acquired 10 minutes after mixing of tau, RNA and 19% glycerol in a 20 mM ammonium acetate buffer at pH 7. (f) Extent of poly(U) induced fibrilization of Δtau187 measured by Thioflavin T assay compared to that of heparin of Δtau187. Pyrimidines can react with ThT and give strong background. For that reason, we used poly(U) to carry out this assay. Error bars show standard deviation from n = 3 in a-b, e-f and the numerical data used are included in (a) Mixing Δtau187 with poly(U) RNA lead to droplet formation (TIFF)Click here for additional data file.S7 Figin vitro were transfected with 8 μg of tRNAPhe -fluorescein (green) per million cells using lipofectamine 2000. The images with tRNAPhe –fluorescein staining are shown together with DAPI (blue) staining of the nuclei. The tRNAPhe-fluorescein and DAPI-stained images show the colocalization of tRNA in the cell. Scale bar is 20 μm.Primary mouse neurons at 14 days (TIFF)Click here for additional data file.S8 FigPipeline of PAR-iCLIP in identifying RNA-tau interaction in intact cells. The protocol of PAR-iCLIP can be found in the Materials and Methods section of the manuscript, as well as in (TIFF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. ThesIt is well known that the addition of a RNA carrier during the RNA isolation procedure increases miRNA recovery, and therefore the subsequent miRNA detection . HoweverAs phenol-based procedures seem to result in poor recovery of miRNAs with low GC content from samples with low RNA abundance , we quanThis is one of the few studies in which miRNA isolation efficiency is compared using different RNA isolation protocols in combination with different RNA carriers ,27. For The research was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Written informed consent was obtained from all individuals, and the study was approved by the Ethical Committee from Hospital Universitario y Politécnico La Fe, Valencia, Spain (#2012/0149).Blood samples were obtained from ten healthy subjects. Platelet poor plasma was obtained by centrifugation at 1500 x g for 30 minutes at 4°C. Collection of platelet poor plasma was stopped 1 cm above the buffy coat to avoid cell contamination. The platelet poor plasma was stored in aliquots at -80°C until further analysis.The quality and isolation efficiency of miRNAs were analyzed in four plasma samples using two protocols: phenol and column based isolation procedures  and column based procedures .The isolation protocols were performed according to manufacturer’s instructions with the following modifications: Plasma samples were thawed in melting ice and centrifuged at 3,000 x g at 4°C for 5 min to avoid the presence of cell debris in the sample. A volume of 200 μl of plasma was transferred to a new microcentrifuge tube as starting material in all the protocols. Then, 1 ml of QIAzol Lysis Reagent for the Q protocol or 60 μl of Lysis Solution BF for the E protocol was added and the mixture was incubated for 5 min at room temperature. Next, 1 μg of RNA carrier  and 1 μl of synthetic miRNA mix  were added in both protocols. Optimum RNA carrier concentration was empirically determined . FinallyAfter the analysis of preliminary results, total RNA from sixteen extra plasma control samples was isolated with Q and E protocols in presence or absence of yeast RNA carrier in order to check the isolation efficiency of small RNAs with different GC content and thermodynamic stability. Thermodynamic stability was evaluated by the free energy of the most stable secondary structure of miRNAs (miRNA ΔG).The RNA concentration and quality were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). We used the A260/280 and A260/230 ratios to check for possible co-purified contaminants during the RNA isolation .RNA concentration and integrity were also analyzed using the Agilent RNA 6000 Nano Kit for total RNA and Agilent Small RNA kit for low molecular weight RNA in the Agilent 2100 Bioanalyzer (Agilent Technologies) .2 μl of RNA were reverse transcribed in duplicate in a final reaction volume of 10 μl using the Universal cDNA Synthesis kit II (Exiqon). This input RNA volume was empirically adjusted . The MasIn the qPCR step, each 10 μl reaction contained 4 μl of cDNA sample (previously diluted 1/40), 5 μl of ExiLENT SYBR® Green master mix (Exiqon) and 1 μl of microRNA LNA™ PCR primer mix (Exiqon). qPCRs were performed following manufacturer's instructions using the LightCycler 480 real-time PCR system (Roche) in 384 well plates, and each sample was amplified in duplicate. After denaturing the cDNA and activating the enzyme polymerase for 10 min at 95°C, the cycling conditions were as follows: 45 cycles consisting of denaturation at 95°C for 10 s and then annealing/extension at 60°C for 60 s (with ramp rate of 1.6°C/s). Cq values, using the second derivative method, and melting curves were obtained using the LightCycler 480 software (Roche).We used serial tenfold dilutions of known concentrations of the synthetic UniSp2 RNA Spike-in (UniSp2) to create a standard curve. The slope of the plot was used to calculate the qPCR efficiency (1).It is unknown whether differences in Cq values between isolation protocols arise from different RNA recovery efficiencies, different inhibitor removal efficiencies, or both. Therefore, we evaluated the qPCR Eff in four samples isolated with the Q and E protocols in the presence or absence of RNA carrier (yeast RNA or MS2 bacteriophage RNA), performing a tenfold serial dilutions experiments of each sample and quantifying the UniSp2. The results of the slope of each sample were also translated into a value for qPCR Eff. When no differences are observed between standard and sample qPCR Eff values, we can assume that qPCR is not influenced by inhibitors, thus differences observed between isolation protocols are caused by different isolation efficiencies (Isol. Eff).The concentration of each sample was calculated by simple interpolation of its Cq into the standard curve, and the Isol. Eff was calculated (2) using the following parameters: “UniSp2 CC” the absolute concentration of UniSp2 recovered calculated by qRT-PCR, and “UniSp2 TMC” the theoretical maximum concentration of UniSp2 that can be recovered.In an initial approach, we evaluated Cq values of the synthetic UniSp2 and UniSp6 RNA spike-in (Exiqon) and the miRNAs hsa-miR-103a-3p and hsa-miR-451a in four plasma-derived RNA samples, isolated with Q and E modified protocols, in presence of yeast RNA (yQ and yE) or MS2 bacteriophage RNA (mQ and mE) as carrier and also without RNA carrier (wQ and wE) . We usedWe evaluated the “fold recovery” (FR) of several miRNAs with different GC content and intra-molecular ΔG in twenty plasma-derived RNA samples isolated with the Q and E modified protocols in presence (y) or absence (w) of yeast RNA carrier . We defined FR as the miRNA mean abundance normalizing by plasma volume and taking as reference the protocols without RNA carrier . It shows us how much miRNA is recovered after carrier addition when it is referred to the same protocol without carrier, for instance, yE referred to wE.This type of normalization can theoretically be performed because identical sample and elution volumes were used in each isolation protocol. The FR for each isolation protocol was calculated using as reference the average of results of the relative quantification using wE or wQ protocols . Finally-ΔΔCt method [We evaluated two normalization strategies in order to acknowledge the most consistent. Thus, we compared differences in “fold abundance” values between different protocols within the same normalization strategy. The normalization strategies were: a) plasma volume normalization  and b) nt method . In bothP-values <0.05 were considered significant.Comparisons of RNA concentration, raw Cq values, miRNA values normalized by plasma volume or by miRNA-103a-3p, were performed using the two-tailed Wilcoxon Signed Ranks Test with the IBM SPSS Statistics 20 software. Correlations were calculated using the two-tailed bivariate Pearson correlation test. Stepwise multiple regression analyses were used to calculate the variables that predict variance in FR. The predictor variables included the miRNA SRA, base 10 logarithm of miRNA SRA, the GC content, and the miRNA ΔG. In this work we compared different modified isolation protocols  named here as E and Q protocols. Furthermore, these isolation protocols were used with two different types of RNA carrier (Torulla yeast RNA carrier and MS2 bacteriophage RNA carrier) and without RNA carrier. After preliminary results we discarded MS2 carrier.The RNA concentrations estimated by spectrometry, determined as a peak at 260 nm, were different between each isolation protocol and RNA carrier employed. The mean values ranged from 9.4 ng/μl obtained by the wQ protocol, to 27.7 ng/μl obtained by the mQ protocol . SimilarThe A260/280 and A260/230 ratios were used to assess the purity of RNA . Ratios Total RNA concentrations obtained with the Agilent 2100 Bioanalyzer were lower than those obtained by spectrometry . The higSimilar qPCR Eff was observed with the standard curve (87%) and the ten-fold diluted samples (85–91%) , indicatThe highest miRNA concentration  was obtained using the yQ protocol in all miRNAs studied compared to the other isolation protocols used , whereasWhen we analyzed the FR values of miRNAs normalized by plasma volume, we observed that the yQ protocol in almost all miRNAs analyzed gave significantly higher FR compared to the other protocols . yE protP<0.001) and the miRNA ΔG  were observed, but no correlation with miRNA SRA or with the logarithm of miRNA SRA were observed. Furthermore, using mean FR values of miRNAs, the correlation with the GC content improved  (2 = 0.703). To reduce the influence of GC content in the miRNAs analyzed , and miRNA ΔG  were observed. When mean FR values for each miRNA were used, the correlation with miRNA SRA was not significant and the correlation with miRNA ΔG increased   . HoweverP<0.001) . On the = 0.002) . This meP<0.001) and the logarithm of miRNA SRA  were observed. These correlations increased when the mean FR values were used  (2 = 0.535). To verify if other variables influence the recovery of the miRNAs and to avoid the influence of very abundant miRNAs in the sample, we removed from the analysis values for UniSp2 spike-in and miR-451a, both overrepresented in the samples, with three and two orders of magnitude above the miRNAs less represented in the sample , the logarithm of miRNA SRA , and miRNA ΔG  were observed. When mean FR values of miRNAs were used, positive correlations with the miRNA SRA and the logarithm of miRNA SRA were observed . The stepwise multiple linear regression analysis without UniSp2 and miR-451a was repeated using mean FR values as dependent variables. In this case, the logarithm of miRNA SRA and the miRNA ΔG were predictors of FR for yQ protocol and the predictive equation obtained was: yQ = 7.294 + 1.892 x logarithm of miRNA SRA—0.365 x miRNA ΔG (adjusted R2 = 0.382). Furthermore, performing correlations using only miRNAs with GC content between 25% and 75% percentile as before, significant correlations between the individual FR values of miRNAs in yQ protocol with the miRNA SRA , the logarithm of miRNA SRA , and miRNA ΔG  were observed. When mean FR values of miRNA were used, significant correlations with miRNA SRA and with the logarithm of miRNA SRA were observed , but the correlation with miRNA ΔG was no significant   . HoweverP<0.001)  or with = 0.054) . This mewww.miRbase.org). Thereby, for instance, using miRNAs with extreme GC content could give rise to models in which ΔG, or relative abundance of miRNAs, or both are less important or negligible.Our data showed that the use of RNA carrier increases the isolation efficiency of miRNAs in a specific way, and this recovery depends, to a greater or lesser extent, of miRNA SRA, ΔG and GC content. Furthermore, this isolation efficiency varies between protocols because miRNA SRA, ΔG and GC content have different weight in each protocol. It is important to highlight that these predictive models obtained are dependent of the miRNAs used. In this work we used miRNAs with a difference of about three orders of magnitude of sample relative abundance (SRA), ΔG between -3.4 and 3.3 Kcal/mol and, GC content between 30 to 64%. However, there are miRNAs with more extreme values, for instance, GC content of human mature miRNAs ranges from 8.70 (hsa-miR-2054) to 95.24% (hsa-miR-1908-3p). These data were calculated from human mature miRNAs sequences available on miRBase 21.0 (P<0.001). As it was mentioned above, these differences are not real because the same twenty plasma samples were used in all isolation protocols. Thus, these differences in fold abundance are apparent fold changes (apFC) and are due to different recovery efficiencies between distinct miRNAs using the same method and furthermore, to different recovery efficiencies for the same miRNA using different isolation methods.Once we analyzed the “fold abundance” of miRNAs normalized by plasma volume . For this purpose, we obtained by qRT-PCR the FR of several miRNAs normalized by plasma volume with different GC content and miRNA ΔG . We obseWe also analyzed the fold abundance of miRNAs normalized by an internal reference miRNA (hsa-miR-103a-3p). This pattern was different to that observed when miRNAs were normalized by plasma volume . SpecifiThus, the RNA isolation protocol employed and the inclusion of RNA carrier are important analytical variables to take into account, together with other pre- and analytical variables, such as type of sample (serum or plasma) –40, antiIn summary, we observed that the use of qRT-PCR is better than the use of absorbance values or electropherogram to compare different RNA isolation protocols. In fact, we can not rule out that results obtained from plasma-derived RNA without carrier on Nanodrop were noise. In the absence of RNA carrier, wE protocol was more efficient than wQ protocol, but in the presence of carrier yQ protocol was more efficient than yE protocol. In our study, yeast RNA carrier renders a more efficient RNA isolationthan MS2 RNA carrier in Q and E modified protocols. Our results point out that the use of yeast RNA as carrier increases the isolation efficiency of miRNAs depending on the relative abundance in the sample, the GC content and ΔG of miRNAs. We also observed that these variables influence differently according to the isolation protocol used. That is, yQ protocol mainly recovered high abundant miRNAs, and yE protocol mainly recovered miRNAs with low GC content and low ΔG and therefore with high intra-molecular thermodynamic stability. In general, we also observed lower variations in fold abundance of miRNAs between protocols when normalizing by an internal reference miRNA instead of normalizing by plasma volume  Tables. S1 FigA) Dilution curve of one plasma-derived RNA sample and isolated with Q modified protocol using different concentrations of yeast RNA carrier (yQ). UniSp2 spike-in was quantified and we observed that from 0.5μg of yeast RNA as carrier did not improve RNA isolation, we also observed that higher carrier amounts did not affect the qPCR reaction. We selected 1μg of yeast RNA as carrier. B) Dilution curve of eight RNA samples derived from plasma and isolated with Q modified protocol with yeast RNA as carrier (yQ) using different input volumes to the RT reaction. UniSp5 spike-in was quantified and we observed that if too much RNA is added to the RT reaction linearity is lost due to the presence of inhibitors of qPCR reaction co-isolated during the sample preparation. We observed that 2μl was the best input volume for the RT-qPCR in our plasma samples.(PDF)Click here for additional data file.S2 FigThe same control sample was isolated using different protocols and RNA carriers.(PDF)Click here for additional data file.S3 FigA) Standard curve from serial dilutions of RT product of UniSp2 of known initial concentration was performed plotting Cq values vs logarithm of UniSp2 concentration (Log Conc). The equation of the straight line and the correlation coefficient (R2) were obtained by linear regression. Using this equation, qPCR efficiency was calculated with the formula: qPCR Eff (%) = 10(1/slope)-1 x 100. Furthermore, we performed serial dilutions of samples (B) to obtain the qPCR efficiency of each protocol. As qPCR efficiencies were similar, Cq values of samples were interpolated to obtain the logarithm of concentration. Once the UniSp2 sample concentration was known the efficiency of recovery of each protocol and carrier combination was calculated with the formula: Isol. Eff (%) = (UniSp2 CC/UniSp2 TMC) x 100. Being “UniSp2 CC” the absolute concentration calculated by qPCR, and “UniSp2 TMC”, the theoretical maximum concentration (40 pM).(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableThe same control sample was isolated using different protocols and RNA carriers.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.