Saliva, as a non-invasive and easily accessible biofluid, has been shown to contain RNA biomarkers for prediction and diagnosis of several diseases. However, systematic analysis done by our group identified two problematic issues not coherently described before: (1) most of the isolated RNA originates from the oral microbiome and (2) the amount of isolated human RNA is comparatively low. The degree of bacterial contamination showed ratios up to 1:900,000, so that only about one out of 900,000 RNA copies was of human origin, but the RNA quality (average RIN 6.7 + /− 0.8) allowed for qRT-PCR. Using 12 saliva samples from healthy donors, we modified the methodology to (1) select only human RNA during cDNA synthesis by aiming at the poly(A)+-tail and (2) introduced a pre-amplification of human RNA before qRT-PCR. Further, the manufacturer’s criteria for successful pre-amplification  had to be replaced by (3) proofing linear pre-amplification for each gene, thus, increasing the number of evaluable samples up to 70.6%. When considering theses three modifications unbiased gene expression analysis on human salivary RNA can be performed. The salivary glands secrete fluid transported from serum as well as tissues surrounding the glands. Other human saliva constituents are from the oral mucosa, periodontium, and the oral microbiome4. Hereby, saliva has been shown to contain RNA biomarkers  for prediction and diagnosis of several diseases5, especially of the oral cavity such as oral cancer7 or disorders of the salivary glands9.During the last two decades, saliva has become of increased interest as an easily accessible and non-invasive source of human biomarkers. Besides DNA, proteins and various metabolites, RNA has also been shown as a promising marker in other tissues and body fluids, providing complex gene expression information. Because saliva is derived from several tissue sources and that it also contains large amounts of total RNA make it one of the most attractive diagnostic, prognostic, and monitoring tools for both systemic and oral diseases11. Like peripheral blood, saliva contains a broad range of hormones, enzymes, antibodies and genetic materials which have been infiltrated from blood through various transport mechanisms such as ultrafiltration, diffusion, paracellular routes and active transport12. It’s a mirror of systemic processes that reflect the levels of endogenous and exogenous substances like drugs. This versatility allows for the application of saliva in the diagnosis not only for salivary gland disorders and oral diseases but also for systemic conditions (e.g. pancreatic biomarkers13).Saliva aggregates information from several bodily sources. Because saliva is a plasma ultrafiltrate, this means most compounds found in blood are also in saliva, leading to the aphorism that saliva is a “mirror of the body”15. For analysing nucleic acids originating from systemic processes, salivary supernatant would be preferable compared to whole saliva as described in some studies16. But isolation of exosomes is laborious, resulting in low RNA yields. As a more robust but—due to the high background—less sensitive alternative, we examined whether whole saliva could be used.While the majority of cellular nucleic acids (DNA and RNA) contained in human saliva originate from the buccal mucosa, cell-free nucleic acids originate from a wide variety of sources within the body, mainly encased in exosomes2 and simplified logistics of sample collection . These features underscore the utility of salivary biosamples as economical and fast tools for diagnostic screening and treatment monitoring  in routine clinical practice. Saliva sampling could be an efficient alternative in an emergency situation such as a large-scale radiological accident or nuclear mass casualty scenario for which an easily accessible biosample could be appropriate for high-throughput biodosimetry19. The great majority of studies dealing with disease biomarkers based on gene expression have been performed using blood or tissue that requires collection by a trained person. With concerted effort, the drawbacks of saliva samples (low RNA yield and high levels of non-human RNA) could be mitigated with methodologic improvements in the laboratory approach.Compared to blood, whole saliva has numerous advantages: non-invasive and simple sample collection (possibly by the study subject or an untrained person), easy and repeated sampling in older people and children24. We recently became interested in isolating RNA from saliva. Contradictory results indicated two major challenges:Overwhelming bacterial contamination of the samples.Low abundance of human RNA compared with blood.Our group has extensive experience in the isolation of RNA from whole blood, its cellular subgroups and biopsies of different tissue sourcesIn this study, we qualitatively and quantitatively describe these two challenges and how we sought to overcome them. A consistent method for performing gene expression analysis in saliva and dealing with these limitations has never been coherently described before and this work may improve efforts of others in trying to extract RNA in a consistent and well documented way. Herein, we focussed on mRNAs which have shown to be promising biomarkers in other types of body tissues and fluids.In order to process human whole saliva to gene expression analysis, we modified several aspects of manufacturer recommendations in the workflow of RNA isolation, cDNA synthesis and Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) in specific ways as described in Table The experimental design was granted by the Ethics committee . The qRT-PCR related measurements  were performed according to the standard operating procedures implemented in our laboratory in 2008 when the Bundeswehr Institute of Radiobiology became DIN-accredited by TÜV Süd München, Germany (DIN EN ISO 9001/2008).Whole saliva samples were collected using 4 ml ORAgeneRNA (RE-100) vial collection kits from DNA Genotek according to the manufacturer’s instructions . The kit is an all-in-one system for the collection, stabilization and transportation of RNA from saliva. Unstimulated whole saliva was collected from twelve healthy donors . The self-collection kits are provided with an RNA stabilizing solution to prevent degradation of RNA by RNases. Two ml of saliva was expectorated into the provided vial and mixed with 2 ml stabilizing solution in the lid, making a total volume of 4 ml. The whole saliva was collected from 9 to 10 am and was preserved in the OrageneRNA Self-Collection Kits after having been shaken vigorously. Samples were stored at room temperature overnight and placed in a freezer (− 20 °C or lower) for storage within 1 day.After completing normal oral hygiene, donors were not allowed to eat or smoke 2 h prior to collection or to drink at least 1 h prior to collection.As a “positive control”, whole blood samples (2.5 ml) from six of the twelve healthy donors were drawn into PAXgene Blood RNA tubes  at the Bundeswehr Institute of Radiobiology on the day of saliva collection. The tubes were gently inverted (10 times), kept at room temperature overnight and then stored at − 20 °C.All samples were anonymized and obtained with informed consent from all donors. Sampling methods were carried out in accordance with the institutional guidelines and regulations.Whole saliva samples were processed following a combination of the ORAgene RNA purification protocol and the mirVana kit protocol . Total RNA, i.e. human RNA and pan-bacterial RNA, was isolated. In brief, the frozen (− 20 °C) ORAgene RNA collection tubes were thawed at room temperature.ORAgene RNA purification protocol25: after vigorous shaking, the samples were heated in a 50 °C water bath for one hour in order to homogenize the solution. After taking aliquots of 500 µl  and incubating them at 90 °C for 15 min, samples were cooled to room temperature and 20 µl ORAgene neutralizer solution  was added to each aliquot as buffer and to precipitate the nucleic acids. After incubation on ice for 10 min and centrifugation at 13,000g  for 3 min, the cell-free clear supernatant was pipetted to a fresh Eppendorf tube, while the separated pellet containing the turbid impurities was discarded.The first steps were performed according to the mirVana kit protocol26. We added the Lysis/Binding Solution from the mirVana kit. Then total RNA including small RNA species was isolated by combining a Phenol–Chloroform RNA precipitation with further processing and purification using a silica membrane. After several washing procedures to purify RNA from other residual debris, DNA ingredients became digested on the membrane . RNA was eluted with 100 µl RNAse free water in a collection tube and the aliquots were pooled for each sample before freezing at − 20 °C. The filters were discarded, and the tubes were stored until quantitative and qualitative analysis.At this step we diverged from the ORAgene RNA purification protocol by skipping the precipitation step with Ethanol and adopted the The PAXGene tubes containing the whole blood (n = 6) were thawed, washed and centrifuged according to the PAXgene Blood RNA system protocol . Cells in the supernatant were lysed , then the Lysis/Binding Solution was added and further steps were performed according to mirVana kit protocol described above.260/A280 ≥ 2.0 (Nanodrop) and a RNA integrity number (RIN) ≥ 6 would indicate adequate quality of the saliva samples for qRT-PCR analysis.Quantity of isolated total RNA from saliva and blood was measured spectrophotometrically using NanoDrop One Microvolume UV–Vis spectrophotometer . The results were displayed in ng/µl along with ratio absorbance value at 260 and 280 nm. The ratio of absorbance at 260 and 280 nm (260/280) was used to assess the purity of RNA in the sample. RNA integrity/quality was assessed by the 2100 Agilent Bioanalyzer  and DNA contamination was controlled by conventional PCR using a β-actin primer. Taking known quantitative and qualitative baselines from RNA specimens isolated from whole blood, a ratio of A27 . Every single strand of isolated RNA was converted into cDNA via reverse transcription using random primers. The amount of RNA input was always determined to 1 µg. The calculated sample input volume was diluted in RNase free water until a final volume of 50 µl was obtained. Total RNA was reverse transcribed in 100 µl of total reaction mixture containing 10 µl of 10 × RT buffer, 4 µl of 10 mM deoxynucleotide triphosphate (dNTP), 10 µl of random primers, 21 µl of RNase free water and, 5 µl of reverse transcriptase enzyme. Each RNA sample was reverse transcribed using a two-step PCR protocol: 2 h and 15 min in a cycle of 25 °C for 10 min, 37 °C for 2 h and 85 °C for 5 min followed by infinite hold at 8 °C.For analyzing gene expression of human rRNA (18S) and pan-bacterial rRNA , total salivary RNA was conventionally converted into complementary DNA (cDNA) via reverse transcription using the High-capacity cDNA reverse transcription kitBecause of the presence of specific abundant transcripts, namely 16S and 18S, the synthesized cDNA was serially diluted using a buffer II . Three different cDNA dilutions were prepared out of the stock cDNA solution: 10 ng/10 µl, 0.1 ng/10 µl and, 0.01 ng/10 µl. The concentration with the least amount of cDNA (0.01 ng/10 µl) was used for qRT-PCR amplification of 16S and 18S. The diluted cDNA samples from the High Capacity Kits were stored in − 20 °C until qRT-PCR.The quantity of synthesized cDNA was measured fluorometrically via Qubit 2.0 Fluorometer  using Qubit ssDNA Assay Kit.20 primers which selectively target and amplify the 3′ poly(A) + tail of the vast majority of eukaryotic messenger RNA (mRNA) and thereby is specific for human RNA only. As a result, pan-bacterial RNA will be excluded from further processing. This decreased non-human RNA amplification bias afterwards which is common in the presence of RNAs of non-human origin.The SuperScript III first-strand synthesis system was newly introduced to synthesize cDNA from the isolated salivary RNA in order to subsequently perform gene expression analysis of human origin. The SuperScript III reverse transcriptase catalyzed reaction utilizes Oligo (dt)28. In order to increase the amount of input for downstream gene expression analysis, our samples were steamed at 45 °C for 90 min followed by elution with 30 µl of RNase free water. Using the concentration from repeated NanoDrop measurements, the amount of RNA input was defined as 0.5 µg, conforming to the maximum input volume of 8 µl. Each reverse transcription reaction contained 1 µl of Annealing Buffer, 1 µl of 50 µM Oligo(dT)20 primer, 10 µl of 2 × First-strand Reaction Mix, 2 µl of SuperScript III/RNaseOUT Enzyme mix and RNase/DNase free water . The ingredients were processed in a 0.2 ml PCR tube according to the manufacturer’s instructions in a two-step PCR. Afterwards, the tubes were cooled on ice and stored at − 20 °C until subsequent pre-amplification. To increase the cDNA output, the reactions were performed twice for each sample and later combined into one tube prior to pre-amplification and qRT-PCR.According to the kit description, the amount of starting material can vary from 1 pg to 5 μg of total RNA and the maximum input volume of RNA is 8 µl29. Based on the manufacturer, multiplex amplification can be performed by pooling up to 100 TaqMan Gene Expression Assays of interest and combined with cDNA and TaqMan PreAmp Master Mix for pre-amplification PCR29. The pre-amplification product was then used for qRT-PCR, using the corresponding TaqMan Gene Expression Assays.To detect low abundant RNA species, pre-amplification was required. We added this step as part of our methodologic improvements  were utilized and pooled to enable the multiplex amplification of specific cDNA targets. Candidate genes were selected based on two criteria: (1) Genes that were known to be expressed in sufficient quantities for gene expression analysis in whole blood and/or (2) genes that were described to be radiation sensitive in previous studies30. To ensure linearity of the pre-amplification step, the thermal cycling was set to 10 and 14 cycles for each sample in order to show linearity and, thus, an unbiased pre-amplification. For instance, a Ct value of 32 before pre-amplification would result in expected Ct values of 22 and 18 after 10 and 14 cycles of pre-amplification, respectively.Pre-amplification was performed according to the TaqMan PreAmp master mix kit protocol. In the present work, four different TaqMan Gene Expression Assays . The qRT-PCR reaction contained TaqMan Universal PCR Master Mix and one of the inventoried TaqMan Gene Expression Assays for separate detection of candidate transcripts. All measurements were run in duplicate. Using a 96-well-forat TaqMan qRT-PCR platform, cards were sealed, centrifuged  and transferred into the ABI PRISM 7900HT sequence detection system. All technical procedures for accurate qRT-PCR were performed in accordance with standard operating procedures implemented in our laboratory in 2008 when the Bundeswehr Institute of Radiobiology became certified according to DIN EN ISO 9001/2008. All chemicals using TaqMan chemistry were provided by Life Technologies, Darmstadt, Germany.Using different sets of primers, two kinds of cDNAs were utilized for qRT-PCR: for human  and pan-bacterial  primer probe designs, cDNA with a low concentration such as 0.01 ng/10 µl from High-capacity cDNA reverse transcription kit was used, whereas for the other primer probe designs ((Ct 18S rRNA – Ct 16S rRNA).Following qRT-PCR amplification, the 18S/16S rRNA ratio was calculated between raw Ct values of 18S and 16S to compare the quantitative relation of human RNA to bacterial RNA. The fold change was determined using the formula: fold change = 2^ Furthermore, the observed and expected fold difference of 18S rRNA between 14 and 10 × pre-amplification as well as 14 ×/10 × and unamplified SuperScript III First-Strand Synthesis SuperMix synthesized cDNA was assessed to evaluate a linear and unbiased pre-amplification among the different samples and genes.True positives, TP (manufacturer predicts success and proof of linear amplification was demonstrated),True negatives, TN (manufacturer predicts failure and no linear amplification was examined),False positives, FP  andFalse negatives, FN .The correspondence of the manufacturer’s prediction of a successful pre-amplification  and our measurements  was examined for each gene and comparison using a two-by-two contingency table comprising:Positive predictive value in percent ),Negative predictive value in percent ),FP in percent (FPx100/(TP + TN + FP + FN)),FN in percent (FNx100/(TP + TN + FP + FN)).To evaluate the predictive ability, we calculated:In this study, a PPV of 100% predicts, that 100% of reactions worked in our measurements when the manufacturer says it should work . A NPV of 100% predicts, that 100% of reactions didn’t work in our measurements when the manufacturer says it shouldn’t work  resulting in an agreement. FP represent the number of samples, in which the pre-amplification should work according to the manufacturer, but our findings (proof of linear pre-amplification) provide insufficient measurements and indicate disagreement. FN represent the number of samples, in which the pre-amplification shouldn’t work according to the manufacturer, but our findings provide reliable measurements, also indicating disagreement.https://www.systatsoftware.com) was used for graphical presentations and comparisons among groups .Excel 2010 (Microsoft) was used for descriptive statistics  and generating the tables. SigmaPlot Version 14  of 6.7 (SD + /− 0.8) was detected for saliva samples using the Agilent bioanalyzer and all saliva samples showed gel-like image bands of 28S and 18S that did not provide any indications for severe degradation . Compared to this, yields of RNA isolated from the 2.5 ml whole blood samples were low  = 549 times less human RNA in saliva compared to blood.Human eukaryotic 18S rRNA raw Ct-values were measured for both saliva and blood samples. Using the same quantity of total RNA  in saliva samples, the mean human 18S rRNA Ct value was 28.1 with large fluctuations . In blood samples, mean 18S rRNA Ct values of 19.0 could be detected with low fluctuations  using the same amount of total RNA per reaction  and 0.47 µg  for cDNA from blood samples . Eukaryotic (18S) rRNA raw Ct-values were measured via qRT-PCR and are graphically shown in supplemental Fig. ΔCt), this resulted in a mean 18S/16S-rRNA-ratio, i.e. degree of bacterial contamination, of 1:110,465. This means that for each copy of a human gene, on average 110,465-times more copies of bacterial genes can be found in the samples, taking again into consideration, that 18S rRNA and 16S rRNA, as the most expressed housekeeping genes, are representative for the majority of human and bacterial RNAs. Depending on the 18S/16S-rRNA-ratio for each sample, the degree of bacterial contamination can range from 42 to more than 900,000 , this strongly suggested that large amounts of salivary RNA isolated originated from bacteria. For this reason, pan-bacterial 16S rRNA raw Ct-values were measured for whole saliva, showing a mean of 17.2  and indicated that there are high yields of bacterial RNA in the sample. The mean ΔCt 18S/16S was 10.9 with a maximum of up to 19.8 . Calculating the fold change between human and bacterial rRNA  were detected in 10 out of 12 the saliva samples after 10 × and 14 × pre-amplification. In two samples (sample ID 11 and 12), no amplification plots could be determined when not pre-amplified and no reliable amplification plots, i.e. Ct values, could be determined when pre-amplified, both after 10 × and 14 × pre-amplification (data not shown). These samples showed the lowest amounts of human RNA and the highest bacterial contamination  − Ct (14X pre-Amp). A ΔCt of 10, respectively 14 indicates 100% linearity and efficiency of the pre-amplification of the target gene. CDKN1A was found to be consistently expressed in all remaining samples with a mean Ct value without pre-amplification of 33.1, 23.2 after 10 × and 19.3 after 14 × pre-amplification  was calculated, resulting in 0.09 for 10 × pre-amplified samples and 0.24 for 14 × pre-amplified samples. Mean ΔCt values are shown in Table CDKN1A. Ct values after pre-amplification were in a range that is totally acceptable for gene expression analysis.After pre-amplification and qRT-qPCR, amplification linearity between unamplified and 10 ×/14 × pre-amplified PCR products of the four genes was calculated by using the following formula: Ct Considering, that according to the manufacturer, a minimum Ct value of 35 is necessary for unbiased pre-amplification, we observed different patterns Table :Figure 4CDKN1A  our findings are consistent with the manufacturer .For FDXR our results were satisfying regarding Ct values after pre-amplification (mean Ct of 23.9 after 14 ×), whereas, with a mean Ct value of 37.4 without pre-amplification and a majority of non-pre-amplified samples being undetermined during qRT-PCR, the results should be biased according to the manufacturer. This shows a high number of false negatives, respectively 70.6% of all comparisons, which indicates that pre-amplification is linear although it shouldn’t work according to the manufacturer. There are again no false positives.In DDB2 the corresponding mean ΔCt (observed vs expected) was 1.8 after 10 × pre-amplification, respectively 2.33 after 14 × pre-amplification .For GAPDH: while the mean raw Ct value without pre-amplification was below the recommended 35 cycles (33.5) compared to DDB2, indicating enough RNA copy numbers for an unbiased pre-amplification and qRT-PCR, the corresponding ΔCt (observed vs expected) was 5.78 after 10 × pre-amplification and 6.69 after 14 × pre-amplification. These outcomes are biased and indicate that the pre-amplification results in an almost 2^6.69 = 103 times difference compared to the expected values. This dysregulation results in a high number of false positives (58.3%), whereas no false negatives were found. Hence, the recommended Ct values of target genes below 35 before pre-amplification proved to be not sufficient of itself and had to be augmented by the criteria of showing a linear (unbiased) pre-amplification.We found a different pattern for Taken together, predictions based on the manufacturer’s criteria are biased and in our examinations can result in as much as 58.3% false positive and up to 70.6% false negative results Fig. . Hence, 12.There are numerous advantages to use human saliva biosamples, as described above. The main benefit is saliva is very non-invasive and easy to access for diagnostic and monitoring approaches. Its potential has been shown in numerous studies examining different salivary biomarkers, e.g. in oral diseases like cancer32, but the method behind has never been explicitly described in detail before, even less described were the challenges, that have to be coped with when working with saliva. Our aim was to provide a robust method to process whole saliva for downstream gene expression studies.There are a couple of studies dealing with gene expression profiling in human salivaSaliva was not a sterile medium compared to blood or other body tissues. Due to the complex contamination of human whole saliva, RNA from this medium is extremely sensitive to rapid degradation of salivary RNA. Endonucleases like RNAses mainly contribute to that. Nevertheless, we showed that, despite these difficult conditions, the RNA integrity is sufficiently well in order to process human salivary RNA for downstream gene expression analysis using the modified workflow. Or in other words: the salivary transcriptome is stable enough.In the course of this study we found that human 18S rRNA abundance compared to blood was several log-fold lower as would be expected considering that the same quantity of total RNA was used. Therefore, we approached details step by step supposing that sufficient amounts of high-quality RNA can be extracted from saliva samples and that there is no inhibition of cDNA synthesis or qRT-PCR Fig. .How to deal with the overwhelming bacterial contamination of the samples?How to deal with the low abundance of human RNA, in order to detect gene expression changes of human origin only?Therefore, two challenges had to be addressed within our effort:20 primers for cDNA synthesis and were able to amplify specific human RNA. But still having to deal with the low yields of material from human eukaryotic origin, downstream pre-amplification became indispensable in the adapted methods we devised to obtain reliable results in downstream gene expression analysis.The bacterial contamination, which had a high inter-individual variance, actually posed the main problem when working with whole saliva. We tried to overcome these problems by focusing on the poly(A)+-tails of human RNA using only Oligo (dt)Even so, it became obvious to us that this might not apply for all genes: according to the manufacturer of the TaqMan PreAmp Master Mix (Applied Biosystems), pre-amplification is uniform when Ct values without pre-amplification are < 35. In order to identify amplification biases, we introduced two rounds of pre-amplification and examined for a linear amplification among unamplified and 10 × and 14 × pre-amplification rounds. Doing that, we identified those 16.7–70.6% of samples which could not be analysed based on the manufacturer’s criteria , but which could be used for analysis following our newly introduced criteria . For GAPDH Ct values < 35 before pre-amplification suggest a different reason, which might be the formation of dimers. Pre-amplification should be linear according to the manufacturer but our findings show 58.3% of false positives. Of note, according to the manufacturer, up to 100 TaqMan Gene Expression Assays can be pooled for specific amplification of these targets and directly compare pre-amplified samples without introducing bias29. This further supports augmentation of the recommended Ct value of target genes below 35 before pre-amplification by the criteria of showing a linear (unbiased) pre-amplification for each target gene.Our findings reliably suggest that pre-amplification achieved excellent results for the majority of samples. But it is not suitable for all genes and an amplification generated bias has to be expected for genes like FDXR). For instance, the suboptimal pre-amplification (∆Ct observed vs expected) in DDB2 (compared to CDKN1A) is most likely due to the fact that Ct values without pre-amplification were already > 35, whereas CDKN1A was < 35 and showed best results (confirming the manufacturer’s specifications). The observed and expected Ct-values for CDKN1A are very close and represent a variance as accepted for technical replicate measurements . These findings suggest that a minimum amount of input is required such that uniformity of pre-amplification should always be checked. In the scenario described above, one solution was to synthesize more cDNA from human RNA with the SuperScript kit in order to increase the input for pre-amplification and qRT-PCR.Nevertheless, the criteria of Ct values < 35 before pre-amplification appeared important but not primarily decisive . On the other hand, pre-amplification doesn’t work in 58.3% of certain gene measurements, although manufacturer predicts success. Furthermore, an improved outcome was achieved if pre-amplification showed a linear (unbiased) relation and that it is necessary to check the linearity.Although the current study was based on a moderate size of samples n = 12) and gene targets n = 4), we performed gene expression analysis for each sample and each of the 4 gene targets without pre-amplification as well as after 10 × pre-amplification and 14 × pre-amplification  in order to show linearity and, thus, an unbiased pre-amplification. This comes up to a total of 144 measurements, respectively 96 comparisons. Regarding the independently performed pre-amplification reactions and following qRT-PCRs, a total of 24 reactions  were performed for each gene  Selective cDNA synthesis of mRNA species of human origin, (2) pre-amplification before qRT-PCR and (3) check uniformity of pre-amplification. These are prerequisites for successful future efforts with respect to processing human RNA for biomarker studies, we believe.For example, 18S rRNA is not synthesized to cDNA when using the SuperScript III First-Strand Synthesis System, because 18S rRNA -transcripts lack a poly(A)+-tail. That’s why 18S rRNA, as a commonly known housekeeping gene, cannot be used as a normalizer in gene expression analysis in the current application. A new housekeeping gene except for rRNA needs to be detected when working on gene expression analysis in human whole saliva.19. Further investigations will show if this poses a limitation of its use as a biomarker.Second, the composition of whole saliva may be influenced by extraneous factors which may limit its use as a biomarker: demographic characteristics , social habits  or oral hygiene are among the factors that can interfere with the composition of saliva at the time of collectionWe decided to use whole saliva as a source for RNA biomarkers instead of salivary supernatant due to the numerous advantages described above. Admittedly, a higher background due to a high amount of non-human RNA is a drawback but using whole saliva is much easier than harvesting supernatant and isolating exosomes. Until now, it is not completely clear, whether the oral microbiome alters gene expression patterns in the salivary glands and to what extend this affects the robustness of salivary transcriptomics as a diagnostic tool, e.g. considering gland disorders. Rather, we chose a robust, less elaborate and high through-put approach with high utility for future studies.In summary, we report a robust modified methodology to process human whole saliva as a non-invasive and easily accessible biofluid for gene expression analysis. These three modifications, described here will be important to overcome the two challenges of an overwhelming bacterial contamination and low abundance of human RNA in the source material for future applications. We suggest that modifications such as ours will render human saliva as a more attractive material for an expanded number of biomarker studies.Supplementary information.


Melatonin, an important hormone secreted by the epiphysis, is a powerful anti-oxidant with a high potential to neutralize medical toxins. The goal of this study was to demonstrate the beneficial effect of melatonin on epididymal sperm and reproductive parameters in mice treated with acetylsalicylic acid (ASA).Thirty-nine SD rats were randomly split into D1 receptor agonist (chloro-APB), D1 receptor antagonist (SCH23390), and saline groups after preparing NB microinjection model. We observed the effect of NB microinjection of SCH23390, chloro-APB, or saline on the period of induction and recovery time of propofol anesthesia and recorded the changes of electroencephalogram (EEG) simultaneously.P<0.05), without affecting the induction of anesthesia (P>0.05); NB microinjection of SCH23390 produced the opposite effects. NB microinjection of saline did not influence the induction or recovery of propofol anesthesia (P>0.05). NB injection of chloro-APB decreased the ratio of δ power and increasedαand β ratios in prefrontal cortex EEG (P<0.05); NB microinjection of SCH23390 increased δ ratio and decreased β ratio (P<0.05); NB microinjection of saline had no significant effect on EEG (P>0.05)NB microinjection of chloro-APB accelerated the recovery from propofol anesthesia (D1 dopamine receptors in NB are involved in modulating the emergence from propofol anesthesia, but not affecting the induction of propofol anesthesia. General anesthetics can reversibly cause loss of consciousness (LOC), but the specific mechanism is unclear. In addition to the effects on the cerebral cortex and thalamus -4, generThe Nucleus Basalis (NB), which is a part of basal forebrain (BF), is important for activation of the hippocampus and neocortex . DamaginAll laboratory processes were approved by Zunyi Medical University . Thirty-nine SD male rats  were purchased from the Army Medical University . Animals were raised and cared for as previously described . Rat models of microinjectionRats received intraperitoneal injection with sodium pentobarbital (50 mg/kg) and were used to prepare the model of NB microinjection, as previously described . A cannuDrugsD1 agonists (chloro-APB) and D1 antagonists  were soluble in saline (0.9%). The infusion cannula and a tubing were linked to a syringe . Drugs were injected using a precision pump . Drugs were injected at 0.25 µl/min for 2 min. Animals were divided into four groups: saline, chloro-APB, SCH23390, and chloro-APB+SCH23390 (n=8). Experimental protocol In behavioral experiment, the observer did not know what kind of drug is microinjected. Before behavioral experiment, rats were placed in a chamber  for acclimation. We tested the loss and recovery of the righting reflex (LORR and RORR) as previously reported , 14, 15.Induction of anesthesiaAfter adapting for 10 min ; t=0 minRecovery from anesthesiaAfter acclimation, anesthesia induction started ; t=0 minEEG analysesEEG waves were obtained and amplified as previously described . Power sHistological localization°C). Blocks (60 µm) including the NB were prepared using a freezing microtome . The sections including NB were placed on a glass slide, then dried and stained as previously described  and perfused with normal saline and then paraformaldehyde (4%). Brain tissue was soaked in paraformaldehyde (12 hr) and dehydrated in sucrose (30%) for 5 days . Data were shown as Mean±SD. LORR and RORR time were analyzed by repeated measures one-way ANOVA with Histological localizationA total of 39 rats were modeled, in which 32 rat cannula sites were located in BF, while 7 rats were not in BF. Each group contained 8 rats. The tissue micrograph of microinjection site was shown in Behavioral resultsP>0.05, n=8, as shown in The LORR time of rats in saline, chloro-APB, SCH23390, and chloro-APB+ SCH23390 groups were 23.31±2.58 min, 26.14±2.76 min, 24.04±2.43 min, and 25.20±2.47 min, respectively, vs 19.71±2.93 min, P<0.05, n=8), while RORR time of rats in the SCH23390 group was markedly prolonged . RORR time of rats in the chloro-APB + SCH23390 group was shorter than that in the SCH23390 group, but longer compared with the chloro-APB group . Infusion of chloro-APB decreased the ratio in the δ band  in PFC, but increased the power ratio in the α band  and β power . However, Infusion of chloro-APB did not affect θ and γ  and decreased the β band , without changing the relative power in the θ, α, and γ , but the degree of change was lower compared with the chloro-APB group . The results demonstrated that infusion of D1 agonist and antagonist not only changed the RORR time of propofol anesthesia, but also induced the EEG changes in PFC. Compared to pre-microinjection, infusion of chloro-APB +SCH23390 into NB decreased the ratio in the General anaesthesia provides analgesia, forgetting, immobility, retardation of autonomic response, and unconsciousness , 18, of Electroencephalogram studies found that propofol induced EEG activity was similar to sleep slow waves and originated from the same cortical area . Therefoet al. found that the orexin pathway regulates the recovery from isoflurane and sevoflurane anesthesia, while not influencing the induction process (The results showed that injection of D1 receptor agonist into NB did not influence anesthesia induction, while accelerating the emergence from propofol anesthesia. In contrast, D1 receptor antagonists delayed the recovery from propofol anesthesia. The traditional view is that the induction and emergence from general anesthesia are opposite processes, but the results of this study do not support this view. Consistent with our findings, Kelz  process . Lesion  process . Our rec process . These sThe behavioral arousal is usually associated with the cortical EEG activation. Therefore, we observed the effect of BF microinjection of D1 agonist and antagonist into NB on cortical EEG. We found that microinjection of D1 receptor agonist significantly decreased δ ratio and increased the ratio of α and β waves. Slow-δ and α oscillations are markers of propofol-induced unconsciousness . SimilarA previous research demonstrated that methylphenidate causes emergence from isoflurane anesthesia  and dropThe following are the limitations of this study. Firstly, this study only observed the effect of dopamine D1 receptors on the induction and emergence from propofol anesthesia, while effects of D2 receptors were not observed. Secondly, although we observed that microinjection of D1 receptor agonists accelerated the emergence from propofol anesthesia, we did not explore the intracellular signaling mechanisms triggered by D1 receptors. D1 dopamine receptors in NB are involved in modulating the emergence from propofol anesthesia, but do not affect the induction of propofol anesthesia.


Sclerotinia sclerotiorum. This virus, named S. sclerotiorum ourmia-like virus 4 (SsOLV4), has a genome 2,982 nt in length with a G-pentamer (GGGGG) at the 5′-terminus and a C-pentamer (CCCCC) at the 3′-terminus. The SsOLV4 genome has only one large putative open reading frame (ORF) predicted with both standard codes and mitochondrial codes and encodes an RNA-dependent RNA polymerase (RdRp). SsOLV4 is closely phylogenetically related to Pyricularia oryzae ourmia-like virus 1, with 42% identity between the RdRp amino acid sequences. We constructed full-length cDNA of SsOLV4 and synthesized RNA in vitro using the T7 RNA polymerase. The synthesized RNA could transfect S. sclerotiorum protoplasts efficiently. We further found that viral RNA could infect mycelia when mixed with PEG buffer. Our study suggests that a novel genus in family Botourmiaviridae should be established for SsOLV4 and other related viruses and demonstrates that one single-stranded RNA segment encoding RdRp is sufficient for ourmia-like viruses in fungi.Recently, an increasing number of ourmia-like viruses have been found in fungi; however, the features of these viruses remain unknown. Here, we report a novel ourmia-like virus isolated from  Colletotrichum acutatum. More recently, Mycoviruses, which reside in fungi, are widespread in all major fungal taxa in nature  and can Narnaviridae have a naked single-stranded RNA (ssRNA) genome and are grouped into two genera, Narnavirus and Mitovirus. Narnaviridae was originally identified in fungi (Chenopodium quinoa mitovirus 1 was confirmed to infect mitochondria of plants (Narnaviridae or Ourmiavirus were identified from insects or protozoa via next-generation sequencing (NGS)  to adapt these ourmia-like viruses (classified into three genera) and existing ourmiaviruses. Interestingly, only one large RNA segment encoding an RNA replicase has been identified in all of these fungal ourmia-like viruses; whether these fungal ourmia-like viruses have other genomic RNA segments remains unknown.Viruses in family in fungi . It is bin fungi , suggestin fungi . Chenopof plants . Recentlng (NGS) , suggestsequence . The firsequence ; then twsequence ; thus, ttructure . Recentltructure . TherefoCryphonectria hypovirus 1  is a devastating pathogen that can infect many economically important crops and cause fatal white mold and soft rot  virus amino acid sequence database using BLASTX. From this database, we identified 47 contigs belonging to nine viruses, including six contigs that could be assembled into one long contig, which was similar to the sequence of Phomopsis longicolla RNA virus 1 . We temporarily named this virus Sclerotinia sclerotiorum ourmia-like virus 4 .Two Both strains PX14A1 and PX14A4 harbor SsOLV4, and strain PX14A4 was chosen for further study. Strain PX14A4 has a slower hyphal growth rate and cannot produce sclerotia on potato dextrose agar (PDA) medium, and it could only induce lesions that were much smaller than those of the virulent strain Ep-1PNA367 (virus free) on the detached rapeseed leaves . The douNarnaviridae and that of the largest segments of ourmiaviruses. The genome of SsOLV4 contains only one large ORF from positions 414 to 2802 based on the universal genetic code or mold genetic code, encoding a protein with 796 aa (89 kDa) (Saccharomyces (Ourmiavirus and Narnavirus (Narnaviridae (including mitovirus) and ourmiavirus.We obtained the 5′- and 3′-terminal sequences of SsOLV4 using the classic RACE technique, and the full-length cDNA sequence of SsOLV4 was confirmed by RT-PCR. The sequence of SsOLV4 is 2,892 bp in length with a 46.5% G+C content. This length is similar to that of the members in family (89 kDa) . The teraromyces . The putrnavirus . The 5′-rnavirus , right. 1.The ORF-encoded protein contained one conserved domain (RdRp). When the full-length SsOLV4 RdRp was analyzed using Position-Specific Iterated BLAST, up to 52% identity to other ourmia-like mycoviruses was detected . FurtherLeviviridae. SsOLV4 was grouped to the newly established family Botourmiaviridae. To date, the virus taxonomy of ICTV (2018b.v2) shows that Botourmiaviridae includes four genera, Botoulivirus, Magoulivirus, Scleroulivirus, and Ourmiavirus /OMVRA as a template, and the transfected strain Ep-1PNA367RA was obtained. All strains Ep-1PNA367P, Ep-1PNA367SPE, and Ep-1PNA367RA were proven to harbor SsOLV4  or three (in strain Ep-1PNAP) nucleotides were alternated compared to the original template cDNA in pMD18-P1 , causingS. sclerotiorum, strains M6, N39P, N69P, and Ep-1PNA367P were dual cultured with the SsOLV4-free strain Ep-1PNA367hyg (labeled with a hygromycin B-resistant gene) side by side on the same PDA plate for 7 days. Then, the hyphal agar disks were taken from the Ep-1PNA367hyg colony and placed on hygromycin-amending PDA plates. The newly developed hygromycin-resistant subcultures  were subjected to SsOLV4 detection by RT-PCR amplification. The result showed that SsOLV4 could be successfully detected in all the newly developed colonies , family Botourmiaviridae includes four approved genera based on the phylogenetic relationship2. Members of the genera  are mycoviruses ; members in genus Ourmiavirus are plant viruses and have segmented genomes   in egments) . Howeveregments) . In thisSaccharomyces 20S RNA narnavirus (NC_004051.1) and Saccharomyces 23S RNA narnavirus (NC_004050.1) .Interestingly, we found that SsOLV4 has a G-pentamer at the 5′-terminus and a C-pentamer at the 3′-terminus. Similar tetramers have been found in 04050.1) . The G t04050.1) . Similar04050.1) . The divRhizoctonia mitovirus 1 and Rhizoctonia M2 virus genomes, no tryptophan amino acid was encoded by UGA codons, indicating viral genomes could be translated into a putative polypeptide using either mitochondrial or universal genetic codons, and the viruses are proved to locate in both the cytoplasm and mitochondria (Narnaviridae.Mitochondria use both UGG and UGA as tryptophan (Try) codons with a few exceptions . Howeverchondria . The RNAchondria , and mitchondria . Althougchondria . We founchondria . This reNarnaviridae. One hypothesis is that the ourmiaviruses have evolved by reassortment of genomic segments of RNA viruses infecting hosts belonging to different eukaryotic kingdoms, in particular, fungi and plants   . This feSclerotinia sclerotiorum strain PX14A1 and PA14A4 was originally isolated from a sclerotium sample on a diseased stem of rapeseed (Brassica napus) plants in Sichuan Province, China; strain Ep-1PNA367 is a virulent strain of S. sclerotiorum and harbors a virus S. sclerotiorum mitovirus 6-A367. Ep-1PNA367hyg is a hygromycin-resistant strain of Ep-1PNA367. All strains and their derivatives were cultured on PDA at 21°C and stored at 4°C.4, 0.1% (w/v) Snailase  and 1% (w/v) lysing enzymes from Trichoderma . After 3 h of digestion at 30°C, the protoplasts were collected by centrifugation for 10 min at 4,000 × g. The protoplast-containing precipitate was resuspended with 1 ml of STC  and diluted with STC to 1 × 104 cells per milliliter (for transfection) or 1 × 102 cells per milliliter (for elimination). Protoplasts were regenerated on RM medium  at 21°C.The protoplast preparation followed the method described previously. Briefly, 7 g of fresh mycelia was put into a 20-ml digestion buffer with 0.8 M MgSOStrain PX14A4 was subcultured three times by picking hyphal tips on an antiviral PDA medium containing 0.5 mg/ml (w/v) of ribavirin and 0.1 mg/ml (w/v) of cycloheximide. Then the subcultured mycelia were placed onto an antiviral PDA plate covered with cellophane and incubated at 21°C for 48 h. The edge of the colony was harvested and used to prepare the protoplast. After the protoplasts were cultured for 24 h, the regenerated colony was poured onto a fresh PDA.The procedure for dsRNA extraction was described previously . BrieflyS. sclerotiorum RNA reagent  according to the manufacturer’s instructions.To extract total RNA, strains PX14A1 and PX14A4 were allowed to grow on top of a cellophane membrane overlying a PDA plate for 3 days, and about 100 mg of fresh mycelia were harvested and ground to fine powder in liquid nitrogen with a mortar and pestle. Total RNA was prepared by using a NI-de novo assembly by Trinity (v2.9.0) . The paired-end sequence reads were trimmed by Trimmomatic (0.39)  and de n(v2.9.0) . Assembl(v2.9.0) . The rea(v2.9.0) . The map(v2.9.0) .The cDNA synthesis and sequencing of dsRNAs were conducted as previously described . Based oTo determine the terminal sequences of SsOLV4, single-primer-dependent amplification following a method previously described was used , and claEscherichia coli strain TOP10 by heat shock. The clones were confirmed by Sanger sequencing. The plasmids pMD18-T7A2 and pMD18-HMA2 had the correct direction and almost the right sequences. A 1.9-kb segment was obtained from the SalI and ApaI digested plasmid pMD18-T7A. A 3.7-kb segment was obtained from SalI and ApaI digested plasmid pMD18-HMA. The two pieces of segment were ligated by T4 DNA ligase (Takara) to obtain the plasmid pMD18-P1, which contains full-length SsOLV4 cDNA with a T7 promoter in the 5′-terminus and a hammerhead ribozyme in the 3′-terminus . Three kinds of DNA were used as templates (BamHI or SpeI). The synthesized viral RNA was used to transfect the S. sclerotiorum protoplasts as previously described . For northern blot, total RNA was denatured for 10 min at 70°C and separated using denatured agarose gel with formaldehyde and then transferred to a positively charged nylon membrane (GE) using alkaline transfer solution . For the dot blot, RNA samples (2 μ μl) were added on the positively charged nylon membrane and dried out. The dried nylon membrane was cross-linked using a UV cross-linker. The hybridization was performed overnight in a glass tube at 55°C. The chemiluminescent signal generation and detection were performed using an Amersham CDP-Star Detection Reagent (GE).A sequence similarity search was performed using BLASTX  against g for 20 min at 4°C to remove mycelial fragment and nuclei. The supernatant was centrifuged at 14,000 × g for 20 min at 4°C. The pellet was resuspended with 2 ml of 20% sucrose (w/v) in TE buffer and centrifuged with sucrose gradient. Each fraction was collected separately and used for extraction of total RNAs. RT-PCR was used to detect the viruses in different fractions.We isolated cytoplasm and mitochondria  followinS. sclerotiorum to rapeseed, SsOLV4-infected and SsOLV4-free strains (Ep-1PNA367 and Ep-1PNA367hyg) and regenerant strains (N39 and N69) were inoculated on leaves of rapeseed following the methods previously described (p = 0.05.To evaluate the effect of SsOLV4 on colony morphology and the virulence of escribed . Each teMN715322.This article contains previously unpublished data. Sequence file of SsOLV4 is available from the NCBI, GenBank Accession No. DJ and QW designed the research. QW, YF, and DJ wrote the manuscript. QW and FM executed the experiments. QW, FM, JC, and JX performed the data and bioinformatics analyses. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with the authors JX and DJ.


Y RNA are a class of small non-coding RNA that are largely conserved. Although their discovery was almost 40 years ago, their function is still under investigation. This is evident in cancer biology, where their role was first studied just a dozen years ago. Since then, only a few contributions were published, mostly scattered across different tumor types and, in some cases, also suffering from methodological limitations. Nonetheless, these sparse data may be used to make some estimations and suggest routes to better understand the role of Y RNA in cancer formation and characterization. Here we summarize the current knowledge about Y RNA in multiple types of cancer, also including a paragraph about tumors that might be included in this list in the future, if more evidence becomes available. The picture arising indicates that Y RNA might be useful in tumor characterization, also relying on non-invasive methods, such as the analysis of the content of extracellular vesicles (EV) that are retrieved from blood plasma and other bodily fluids. Due to the established role of Y RNA in DNA replication, it is possible to hypothesize their therapeutic targeting to inhibit cell proliferation in oncological patients. RNY1, RNY3, RNY4, and RNY5) encode for Y RNA; they are clustered on chromosome 7q36.1 [The discovery of Y RNA goes back to 1981 ,3 and SSThe Y RNA family is not limited to the transcripts of the four canonical genes described above; there are other 966 hY RNA pseudogenes  scattered on all human chromosomes ). IntereY RNA are conserved molecules ,13,14,15Despite their relatively short length, Y RNA have a complex 3-D structure, including both double-helix rigid stems and single-strand flexible loops ; their sTo date, several proteins have been identified that directly interact with either the entire Y RNA or Y RNA-derived fragments; the current knowledge about these interactions is summarized in A special consideration, among Y RNA interacting proteins, should be given to RO60. This protein is a ring-shaped polypeptide  that speThe role of—at least some—Y RNA in the initiation of DNA replication and cell cycle progression is well established. RNA-mediated depletion (RNAi) has been successfully used to knock down the intracellular amount of hY1, hY3, and hY4, demonstrating that this treatment on any of those is sufficient to halt or strongly reduce both processes, while the artificial re-expression of any of them in the same cells is sufficient to restore a pre-treatment situation ,52,53,54The different results obtained for hY5 (no effect on DNA replication upon RNAi treatment) may be explained in at least two ways. Some authors hypothesize that this Y RNA is just refractory to RNA-mediated depletion ,52. AlteThe role of Y RNA in promoting the initiation of DNA replication is in good agreement with their overexpression in various human solid tumors  see als.During the apoptosis, the RNA component of Ro RNP is partly degraded, generating the Y RNA-derived small RNAs (YsRNA); however, Dicer is not involved in their formation, thus their origin and function is not related to those of micro-RNAs ,60. ThesMKI67 gene), a 359-kD nuclear protein commonly used to detect and quantify proliferating cells, with increased expression associated with cell growth and absent only during the G0 phase of the cell cycle, i.e., in mitotically quiescent cells. Instead, expression levels for each of the four hY RNA were normalized to HPRT1 mRNA; HPRT1 gene encodes hypoxanthine phosphoribosyltransferase and is involved in the generation of purine nucleotides through the purine salvage pathway. HPRT1 shows very low variation in expression levels between different human tissues and cell types. The choice of these genes as a reference has been questioned later in works on prostate and bladder cancers (see below) and, for this reason, some authors hypothesize that the results obtained by Christov and co-workers might not be fully reliable, at least in those two tumors. (ii) Christov and collaborators do not distinguish different subtypes of cancer samples, and this might explain the only partial overlap of the results on Y RNA expression, for example, in kidney or lung cancers. Indeed, it is possible to identify specific Y RNA signatures in different cell types, thus this is not a trivial point. (iii) The numbers of tumor and control samples are low and, despite the statistical analysis, the possibility to introduce errors is high. In particular, the specimen sizes were as follows, where the first number in parentheses indicates the number of samples of normal tissue and the second indicates the number of samples of cancer tissue: bladder ; cervix , colon ; kidney ; lung ; prostate . (iv) In this work, there is no distinction between the intracellular and extracellular—either free or embedded inside EV—amount of Y RNA. Additionally, this point has been discussed in subsequent works, indicating that sometimes differences of Y RNA expression in these two environments are significant and might underline a specific excretion mechanism of these molecules in some cancer types , circular RNA (circRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), etc. . In a fiRNY1, RNY3, and RNY4 expression being highly correlated to each other, whereas RNY5 expression levels are less distinctly correlated with the other three. These authors noted that the low abundance of hY1, hY3, and hY4 is typical of muscle-invasive BC (MIBC) compared to non-muscle-invasive BC (NMIBC), whereas hY5 levels in those BCs were comparable. Moreover, the low amount of hY1, hY3, and hY4 also correlates with lymph node metastases and advanced grade and, consequently, with patients’ overall  and cancer-specific  survival in a univariate (but not multivariate) analysis. No correlation was found with age or gender. The striking difference found in the expression level of Y RNA between the two available studies on BC might be explained in at least three ways. First, by the much lower number of samples (n = 4 vs. n = 88) that might have limited the reliability of the results shown in the first report. Second, by the different reference gene used in the earlier work, here substituted by the small nucleolar RNA SNORD43 and the U6 snRNA RNU6-2, which are considered suitable reference genes for urological malignancies. Third, by the relative abundance of specific cell cycle stages in the samples, since Y RNA are more abundant during DNA replication and less during mitosis (see above). An increased cell proliferation might suggest an increased Y RNA expression for promoting DNA replication in cancer cells. However, and quite interestingly, a reduced Y RNA expression has been found also in other cancer types . On the other hand, some limitations affect the work of Tolkach and collaborators as well, such as sample age, RNA integrity, or minor contaminations during sample preparation. In conclusion, further studies are needed to assess with higher confidence if, in BC, Y RNA are downregulated, as the more recent study suggests.In 2017 Tolkach and collaborators reported that all Y RNA are downregulated in BC  , with RNn = 5) and normal controls (n = 5) for the presence and relative abundance of these molecules, finding that a significant proportion (38%) of the identified RNA fragments are indeed Y RNA derivatives (coming from both genes and pseudogenes), with the following subdivision: a major population of 30–33 nt fragments mostly derived from their 5′ end, and a minor population of 25–29 nt fragments almost exclusively derived from their 3′ end. Interestingly, they showed that in breast cancer patients there is both an increase and a decrease of these fragments, but the increase involves mostly 3′ fragments (19/20) and the decrease involves only 5′ fragments (5/5); this occurs despite the fact that 5′ fragments are, in absolute terms, much more abundant in blood serum than their 3′ counterparts (ratio: ca. 20:1). To further validate these data, the same approach was used on 42 additional datasets available from external sources, which revealed a proportion of Y RNA fragments of 13% of the total RNA. Additionally, in these datasets, specific Y RNA fragments either increased or decreased their abundance compared to controls, depending on the BrC diagnosis or tumor characteristics. This result is in good agreement with a previous one describing an enrichment of 3′-end fragments—derived from human hY5—detected in MCF 7 (mammary adenocarcinoma) cells [In 2010 Meiri and collaborators found the upregulation of hY1 and hY3 in this cancer . After ta) cells .RNY1, RNY5 and, above all, RNY4 expression. In addition, Tosar and collaborators showed that 5’ hY4-derived fragments of 31–33 nt are greatly and significantly enriched in the extracellular space of cultured breast cancer cells, suggesting that such fragments are specifically excreted by these cells [More recently , a studyse cells . SimilarGlioma is the most common tumor of the brain, comprising about 30% of all brain tumors and 80% of all malignant brain tumors. To date, only one paper was published on glioma and Y RNA expression . In thisTo date, two reports have been published about the Y RNA quantification in cervix cancer ,62, bothEven in the case of colon cancer, the work of Christov and collaborators is still the main reference . It showIn 2015 Martinez and collaborators reported that Y RNA-derived small RNAs are significantly deregulated in the sera of head and neck squamous cell carcinoma (HNSCC) patients . These pA recent work from Dhahbi and coworkers explored the presence of Y RNA fragments in oral squamous cell carcinoma (OSCC), a form of HNSCC and the most common type of head and neck cancer . In addiBernatsky and collaborators demonstrated that many cancer types, and hematologic malignancies in particular, are substantially increased in patients affected by systemic lupus erythematosus . ChakrabMore recently, Haderk and collaborators studied the role of tumor cell-derived exosomes in the crosstalk with monocytes in patients affected by chronic lymphocytic leukemia (CLL) . They foNotably, both these works suggest that sncRNA-loaded EV play a pivotal role in leukemia cell survival through the creation of a favorable microenvironment. Moreover, they also show that Y RNA (together with other sncRNA) might potentially act on physically distant, normal cells using EV as shuttles, similar to what has been proposed also for Kaposi’s sarcoma (see below).RNY3 and RNY4 is significantly increased in ccRCC samples; moreover, the expression levels of RNY4 alone is inversely correlated with the ccRCC stage and the presence of lymph node metastases . Nonetheless, the study failed to find clear differences in the blood serum of patients vs. controls for any Y RNA; this implies that in this case, Y RNA could be potential tumor markers only when directly using cancer samples. These data are in overall good agreement with those reported before [In 2008, Christov and collaborators found Y RNA overexpression in their kidney cancer samples, especially for hY1, hY3, and hY4 , but in d before  for hY3,d before , normal d before ). Howeved before ) and thet translocation, constitutively expressing the NPM-ALK fusion protein [RNY4 gene, and the highly similar pseudogenes RNY4P7, RNY4P10, and RNY4P20. The most abundant fragment includes the first 32 nt at the 5′-end of RNY4 transcript and includes the entire stem region and part of the loop. The result was not confirmed by qRT-PCR for the fragment, but was validated for the full length hY4. In these patients, the fragment can be found both as free RNA and inside exosomes, while the full length hY4 is specifically enriched in exosomes of ALCL patients; the upregulation of full length hY4 has been further confirmed in an extended cohort of 44 ALCL samples and 19 controls. The extended cohort was also used to correlate hY4 presence and disease aggressiveness, revealing that the full length hY4 was more abundant in exosomes of ALCL patients with advanced disease stages (stage 3–4 vs. stage 1–2); among these patients, those relapsing had a higher amount of this RNA compared to those with stable, complete remission. Taken together, these data support the quantification of hY4 as a potential biomarker for both tumor identification and staging, through a non-invasive analysis (liquid biopsy), an important aspect in children management.Anaplastic large cell lymphoma (ALCL) is a fast-growing non-Hodgkin lymphoma (NHL) and accounts for 10–15% of pediatric and adolescent NHL; ALCL in pediatric patients is almost always ALK-positive and mutant cells show the  protein . A recen protein . The autLung cancer (LC) is the malignancy with the highest incidence for both sexes worldwide. Data available for this type of cancer and Y RNA expression come from three studies. The first is the abovementioned work of Christov et al. , which rRecently, one additional report specifically investigated the link between Y RNA and LC . In thisRNY1, RNY3, and RNY4; average: 26.1% of reads), 30 Y RNA pseudogenes (average: 48.4% of reads), and 194 Rfam predicted Y RNA sequences (average: 25.5% of reads), plus other sequences in traces. The presence of the RNY4 gene and RNY4P pseudogene sequences strongly characterizes these EV, accounting for more than 98% of their respective Y RNA. The authors also related the presence of these Y RNA with the disease stage, finding that five of them  are differentially expressed; RNY3P1, RNY4P1, and RNY4P25 fragments show a specific enrichment in stage 0 samples, compared to control samples or stage I/II samples . The discrepancy between the two works might be due to the different source of EV: directly released by melanoma cell cultures in the first, retrieved by plasma of patients in the second. In conclusion, also in melanoma cells, the relative amount of Y RNA can potentially be a good marker of this disease and, possibly, also of its stage.Lunavat and co-workers analyzed the ncRNA content of EV of the melanoma cell line MML-1 . Addition = 56), normal (n = 36), and benign prostatic hyperplasia  samples. Similar to what found for BC (see above), also here all four Y RNA seem to be 2–4-fold lower than in normal tissue. Moreover, the data obtained show that hY4 and hY5 amounts are significantly lower in PCA compared to both control and BPH specimens, while hY1 and hY3 show this reduction as a trend. Interestingly, expression levels of all Y RNA were similar in both BPH and normal prostate tissue. The authors also found that RNY1, RNY3, and RNY4 expression levels were highly correlated, while RNY5 expression was less distinctly correlated with the other three. In addition, they also found that higher RNY5 expression was associated with poor prognosis measured as biochemical recurrence-free survival. These results are in contrast with those for BC, previously described by the same group [RNY1, RNY3, and RNY4 are highly correlated to the outcome and RNY5 lacks any association. This finding further supports the use of Y RNA as a biomarker for cancer characterization. Additionally, for prostate cancer, these data do not match those reported before [Prostate cancer is the most common malignancy in men. The work of Christov and collaborators showed that, in prostate cancer, hY1 and hY3 are highly abundant, while hY4 and hY5 levels are comparable to controls . These dme group  . Several changes in the total sncRNA profile were detected according to the patient health status and to the ongoing infection(s). The study revealed that, in plasma exosomes, Y RNA are significantly over-represented in all individuals, accounting on average for 93% of the total mapped reads, independently of the HHV8, HIV, and plasmodium infection status. As in other cancers, only 5’-ends-derived fragments were detected, mostly belonging to hY4  and, to a lesser extent, to hY1 (1.8%), hY5 (1.4%), and hY3 (<0.1%). The authors suggest that malaria might induce the release from erythrocytes of exosomes  is a multi-organ cancer that can form masses in the skin, lymph nodes, lungs, gastrointestinal tract, and other organs. It is caused by human herpesvirus 8  and primarily affects individuals with poor immune function. The endemic form, affecting also young people, and its relation with Y RNA expression, has been recently studied by Ikoma and collaborators  in Ugandescribed ) that coescribed . If thesAs reported above, autoimmune diseases cause systemic autoimmune chronic inflammation, also when RO60 is the target. Over the years, evidence has accumulated of a direct relation between tumorigenesis and alterations of the RO60 function, as well as between tumorigenesis and inflammation . Although a direct role of Y RNA has not been recognized in these cases, the strong interaction of these molecules (RO60 and Y RNA) might help understand their role in at least some of these pathologies. For these reasons, we briefly report here what is the current knowledge on this topic.It has been repeatedly shown that patients affected by autoimmune RO60-dependent conditions have a higher frequency of malignancies, including melanoma, T-cell lymphoma, non-Hodgkin lymphoma, and breast carcinoma ,94,95,96Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with an extremely poor prognosis. The work of Liu and collaborators  shows thData about Y RNA biology and, more specifically, about their role in cancer etiology, began to accumulate in the last years. Despite the sparse and sometimes contradictory data, a role for these molecules in cancer formation is emerging. The interaction with several proteins involved in gene expression , the rol


MED12, HMGA2, or FH. Leiomyomas are still widely regarded as a single entity, although early observations suggest that different subtypes behave differently, in terms of both clinical outcomes and therapeutic requirements. The majority of classification studies on leiomyomas have been performed using fresh frozen tissue. Archival formalin-fixed paraffin-embedded (FFPE) tissue represents an invaluable source of biological material that can be studied retrospectively. Methods capable of generating high-quality data from FFPE material are in high demand. Here, we show that 3′RNA sequencing can accurately classify leiomyomas that have been stored as FFPE tissue in hospital archives for years. A targeted 3′RNA sequencing panel could provide researchers and clinicians with a cost-effective and scalable diagnostic tool for classifying smooth muscle tumors.Uterine leiomyomas are benign smooth muscle tumors affecting millions of women globally. On a molecular level, leiomyomas can be classified into three main subtypes, each characterized by mutations affecting either MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3′RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3′RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3′RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in  Uterine leiomyomas, or fibroids, are benign tumors originating from the smooth muscle cells of the myometrium. Leiomyomas are one of the most common human neoplasms, affecting 70% of women of reproductive age . AlthougMED12), a chromosomal aberration resulting in significant overexpression of high mobility group AT-hook 2 (HMGA2), or biallelic loss of fumarate hydratase (FH). Leiomyomas with FH deficiency constitutes a rare but clinically relevant subtype, as pathogenic germline variants in FH predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). This syndrome is characterized by an early onset of multiple symptomatic uterine leiomyomas as well as renal cell cancer and cutaneous leiomyomas . The. The6]. HMGA1 overexpressing leiomyomas and eight myometrium samples from the discovery dataset of George et al. [FH were evaluated further using BasePlayer [Raw whole-exome sequencing (WES) data (FASTQ) of two e et al.  were rete et al. . Data pre et al. . In briee et al. , duplicae et al. . SomaticsePlayer . SomaticsePlayer .This proof-of-principle study demonstrates that 3′RNA sequencing is highly suitable for analyzing and classifying archival smooth muscle tissue material. Future work utilizing this method will not only provide insights into underlying defects of the remaining uncharacterized leiomyomas, but also allow for detection of biomarkers that can differentiate leiomyomas from their malignant counterparts: leiomyosarcomas. 3′RNA sequencing may provide researchers with an accurate tool to retrospectively study the transcriptome of rare tumor subtypes and cancers that are difficult to obtain. To conclude, our observations indicate that a targeted 3′RNA sequencing panel could provide researchers and clinicians with a cost-effective and scalable diagnostic tool for stratifying smooth muscle tumors. Millions of women suffer from leiomyomas, and the ability to accurately stratify each lesion should pave the way towards personalized treatments.


Despite barrier measures and physical distancing tailored by the populations worldwide, coronavirus continues to spread causing severe health and social-economic problems. Therefore, researchers are focusing on developing efficient detection and therapeutic platforms for SARS-CoV2. In this context, various biotechnologies, based on novel molecules targeting the virus with high specificity and affinity, have been described. In parallel, new approaches exploring nanotechnology have been proposed for enhancing treatments and diagnosis. We discuss in the first part of this review paper, the different biosensing and rapid tests based on antibodies, nucleic acids and peptide probes described since the beginning of the pandemic. Furthermore, given their numerous advantages, the contribution of nanotechnologies is also highlighted. To date, six human coronaviruses have been identified: α-coronaviruses , β-coronaviruses , severe acute respiratory syndrome-CoV (SARS-CoV), and Middle East respiratory syndrome-CoV (MERS-CoV) [Different measures have been imposed by authorities, including wearing masks and physical distancing, aiming to stop the virus spread. However, the adopted procedures are not sufficient, and mass screening of coronavirus is more than a necessity. At present, real-time RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) is the most used technique for COVID-19 testing. It is based on specific primers targeting sequences encoding the proteins E, N, S, ORF1b(Open Reading Frame 1b), and RdRp(RNA-dependant RNA polymerase)or a combination of different targets ,5. The mApart from familiar technologies, there have been several reports describing the identification of new biorecognition elements that have been employed in different biosensing schemes ,11. In aSeveral research groups are working on the development of rapid and sensitive assays and biosensors to overcome the limitations of traditional techniques used in coronaviruses detection. These techniques are mainly based on antibodies and nucleic acids including aptamers, clustered regularly interspaced short palindromic repeats (CRISPR), and antisense oligonucleotides.Currently, immunoassays are the most popular diagnostic tools available in the market and used in medical structures. Basically, these methods use antibodies as bioreceptors targeting capsid proteins or whole viruses. In serological testing, capsid proteins are used as viral antigens to bind the immunoglobulins generated by the patient against the pathogen. Antibodies are usually obtained from animal immunization with N, S, or E protein or from the blood samples of patients who are infected . In addiElectrochemical immunosensing is based on the conversion of the antibody–antigen interaction into electrochemical signals. In contrast to traditional immunoassays, innovative transducers allow a highly sensitive quantification of the targeted antigen/antibody in different ways . It shouField-effect transistor(FET)-based biosensing is characterized by many advantages, including sensitivity, fast response, and low-noise detection . MoreoveBesides electrochemical methods, optical biosensors have been widely used in clinical diagnosis and drug discovery due to their analytical performance in detecting biological systems . DiffereSurface plasmon resonance-based biosensors are based on the monitoring of refractive index variations after the formation of the target–receptor complex on the sensing surface. Plasmonic biosensing offers many advantages, including label-free and real-time monitoring . Yong’s Besides label-free sensors, optical sensors, based on chemiluminescent and colorimetric labeling, have been reported for COVID-19 diagnosis. First, Cai et al. developed a chemiluminescent enzymatic serological immunoassay based on a peptide from the S protein. The latter was screened from peptide antigens synthesized from the genomic sequence from GenBank (NC_045512.1). Streptavidin-functionalized magnetic beads were used as an immobilization support for the biotinylated peptide. The binding between IgM and IgG present in serum samples and the magnetic beads was carried out in solution. After binding and washing, the conjugate was reacted with the substrate, generating a luminescent signal, which was measured by a luminometer. In this work, no clinical application was carried out . AnotherDespite the importance of IgGs in detecting previous COVID-19 infection and the association of IgM to recent contamination, the role of secretory immunoglobulins IgA should not be neglected. IgAs(Immunoglobulin A)play a key role in the immune exclusion process limiting the access of microorganisms to the mucosal barriers. It has been demonstrated that elevated total IgA and IgA-aPL(IgA-antiphospholipid) are significantly associated with severe COVID-19 infection ,33. RodaThe different immunosensing strategies described above are summarized in As coronaviruses are positive single-stranded RNA viruses, their detection is usually based on nucleic acid techniques, mainly real-time PCR. However, this technology requires expensive reagents and equipment, in addition to qualified manipulators. Therefore, researchers are focusing on new techniques based on clustered regularly interspaced short palindromic repeats (CRISPR), aptamers, and nucleic acidhybridization to reduce the dependence on RT-PCR.After the identification of CRISPR-Cas systems targeting RNA, numerous studies have shown the potential use of this tool in the detection of different viruses such as dengue and Zika viruses . Cas13 eThe first designed and most used platform, called SHERLOCK showed a successful application for identifying cell-free tumor DNA mutations, genotyping human DNA, detectingdengue and Zika viruses, and distinguishing pathogenic bacteria . SeveralBy combining SHERLOCK with the HUDSON protocol; a process for lysis of viral particles and inactivation of DNases and RNases in body fluids with heat and chemical treatment, no prior RNA extraction is needed . In a reApart from Cas-13, Cas-12 effectors have been also employed for coronavirus diagnosis. For that, CRISPR-Cas12 was combined to DETCTR platform with the DNA endonuclease-targeted CRISPR trans reporter (DETCTR) platform. Extracted RNA is first retro-transcripted to DNA, followed by isothermal amplification using the RT-RPA enzyme. The Cas12a-gRNA complex is then activated through identification of a specific sequence in the amplified DNA, leading to the cleavage of reporters as it was described for the SHERLOCK platform. Based on this principle, different fluorescent lateral flow assays have been developed and tested in clinical samples, showing excellent results .CRISPR-Cas9 effectors have been also exploited to construct a triple line lateral flow assay for the simultaneous dual gene (E and Orf1ab) detection of SARS-Cov2. The assay is based on the recognition between the Cas9/sgRNA and DNA-AuNPs probes. After binding, the formed complex accumulates on the test line and generates a visible colorimetric signal. The originality of this strip is the simultaneous detection of more than one gene which is not available by using Cas13 and 12 requiring nonspecific cleavage .Finally, these studies confirm that CRISPR technology holds a great potential for the diagnosis of the novel coronavirus. It constitutes a promising alternative for the RT-qPCR technique, mainly due to its low cost, simple fabrication and sensitivity.Aptamers are a class of bio-inspired receptors composed of single stranded DNA or RNA. They are selected by a combinatorial process called selection of ligands by exponantial enrichment (SELEX)for their affinity and specificity to a target . They arIn April 2020, Song et al selected the first aptamer specific for receptor binding domain of the SARS-CoV-2 spike glycoprotein. SELEX was carried out by incubating a ssDNA (single stranded DNA) library comprising 40 random nucleotides with RBD protein immobilized on A-beads. After a total of twelve rounds of selection and amplification, two sequences have been chosen. They exhibited a high affinity for the novel coronavirus with respective Kd values of 5.8 and 19.9 nM. In addition to the experimental competition, molecular dynamic simulations suggested that the selected aptamers bind to identical amino acids of RBD, thus providing a promising issue for coronavirus diagnosis, prevention and treatment . The feaApart from the spike protein, Nucleocapsid protein (NP) has been also targeted by SELEX technology. Zhang et al selected four aptamers specific for N protein with high affinity lower than 5 nM.For that, a total of five rounds of selection were performed starting from a library of 76 nucleotides comprising a randomized sequence of 36 nucleotides. After being selected, aptamers were employed in a sandwich-type assay based on four aptamer pairs, which form a ternary complex with NP. Combined with antibodies, this principle was first applied in an enzyme linked immunosorbent assay showing a high sensitivity. Then, an immunochromatographic strip was developed using gold nanoparticles to allow the visual detection of NP in urine and serum . The selBesides to aptamers specifically selected for the novel coronavirus, the potential use of SARS-CoV-1 specific aptamers has been also investigated. Based on the fact that the sequence of SARS-CoV-2 N protein exhibits a homology of 91% with that of SARS-CoV, Chen et al studied the binding capacities of the SARS-CoV aptamer to detect the new coronavirus. Enzyme-linked aptamer binding assay (ELAA) demonstrated that the three tested aptamers exhibit a binding affinity for the SARS-CoV-2 N protein opening the way for their application in COVID-19 diagnosis and treatment. Aiming to validate that, the binding efficiency of aptamer 2 was compared to that of a commercial antibody using Western blot showing similar results. These aptamers could thus be a good alternative for antibodies and immunoassays, currently used for coronavirus detection. Moreover, the use of these aptamers should provide a low cost and rapid solution by avoiding the selection of new candidates specific to SARS-CoV-2 .Recently, an interesting study was published by exploring the binding affinity of a nucleocapsid protein specific aptamer and proximity ligation strategy. The principle is based on targeting the antigenic protein with two different aptamers in order to bring the ligation DNA region into close proximity and initiate ligation-dependent qPCR(quantitative PCR) amplification . The proDespite the excellent characteristics of aptamers, few research reports explored their advantages in COVID-19 diagnosis. Based on the excellent results of the aptasensors discussed above, more techniques will be certainly described in the future by exploring the different sequences selected either for SARS-CoV or SARS-CoV-2.Based on the predictable and specific hybridization of complementary bases, DNA-RNA hybridization has been widely used in RT-PCR and other biomedical diagnosis techniques. Hybridization is mainly dependent on melting temperature; complementary strands can hybridize with each other when the temperature is slightly lower than their melting temperature, while a single mismatch can cause the melting temperature to decrease significantly . Qui et Owing to their simple synthesis and low cost, antisense oligonucleotides could be more advantageous than the other bioreceptors discussed in this review including aptamers and antibodies.In recent years, nanotechnology and nanoengineering have shown a remarkable progress. Nanomaterials have been widely studied and applied in numerous fields including medicine, showing great potential in the diagnosis, prevention, control and treatment of many diseases ,85. ThisAfter discussing the different COVID-19 biosensing platforms integrating nanomaterials in the first part of this review, we focus in this part on the different therapeutic strategies based on nanotechnology. Basically, nanoparticles can be used as drug carriers and viral inhibitors. Moreover, they hold a great potential in vaccine research for the novel coronavirus.Nanomaterials have been reported as excellent carriers for delivering drugs, with high selectivity, to targeted locations in organism. It has been demonstrated that nanocarriers reduce therapeutic agent toxicity, immunogenicity and side effects while increasing drug efficacy. Moreover, certain nanomaterials have been already approved by the FDA for drug delivery such as polylactic-co-glycolic-acid and lipid nanoparticle systems . In vaccine design, nanocarriers constitute a key element either in antigen encapsulation and presentation or boosting of the immune system via the co-delivery of antigen and adjuvant . The abiBesides to vaccine delivery, nanomaterials could be used as antiviral carriers for SARS-CoV-2 treatment. For instance, Ullah et al. proposed a siRNA-based antiviral strategy by using cationic liposomes as a delivery system. Aiming to target ACE2 receptors, the authors proposed an aerosol formulation for intranasal or intratracheal administration. This targeting can be achieved by functionalizing lipid/polymer nanocarriers with highly specific receptors such as antibodies or aptamers. Such a strategy could promote the use of siRNA for silencing and knockdown of viral genes .2, SiO2…) have been used as antivirals against different viruses including hepatitis B, H3N1 and H1N1viruses [Nanomaterials can be successfully conjugated to various functional groups, linkers and bioactive molecules. Moreover, it has been demonstrated that nanoparticles are able to cross the blood-brain and blood-air barriers as well as lymphatic system ,95. Furt1viruses . For ins1viruses .Based on these findings, nanomaterials-based antiviral strategy could be used as promising tool for SARS-CoV-2 inhibition. In the literature, different reviews have discussed the potential of nanotechnology in combating COVID-19 infection ,99. In tThe health crisis caused by the COVID-19 pandemic showed the key role of biotechnologies in biomedical research. As COVID-19 patients display nonspecific symptoms, efficient diagnostic approaches are highly required. Many efforts have been devoted worldwide to meet the urgent need for cost-effective mass screening. In this context, a large array of biosensors has been developed based on different bioreceptors and detection techniques. Many research groups have investigated immunochemical techniques based on antibodies, peptides and aptamers, combined with electrochemical and optical transducers for the sensitive and specific detection of viral antigens and/or immunoglobulins. In parallel, viral genes have also been targeted by using different types of nucleic acids including CRISPR and antisense oligonucleotides. Most part of the developed biosensors was portable allowing rapid, cheap and easy to use point of care testing of SARS-CoV-2. Moreover, some techniques allow the sensitive determination of more than one indicator providing a complete diagnosis and monitoring platform. Furthermore, dual platforms combining simultaneous sample collection and antigen sensing have also been reported. Finally, the selectivity of these technologies has been demonstrated against different types of human pathogens. Besides diagnosis, vaccination is also of a great importance in preventing the propagation of viruses, in particular, with the fast spreading UK virus variant which is raising alarms . In this