These methylations occur in different RNA types, and their distribution is species-specific. 5-methylcytosine (m5C) has been found in mRNA, rRNA and tRNA of representative organisms from all kinds of species. As reversible epigenetic modifications, m5C modifications of RNA affect the fate of the modified RNA molecules and play important roles in various biological processes including RNA stability control, protein synthesis, and transcriptional regulation. Furthermore, accumulative evidence also implicates the role of RNA m5C in tumorigenesis. Here, we review the latest progresses in the biological roles of m5C modifications and how it is regulated by corresponding “writers”, “readers” and “erasers” proteins, as well as the potential molecular mechanism in tumorigenesis and cancer immunotherapy.Epigenetics including DNA and RNA modifications have always been the hotspot field of life sciences in the post-genome era. Since the first mapping of N6-methyladenosine (m Compare7G) Fig. . They in5C) can be traced back to the 1950s, earlier than the discovery of the double helix structure of DNA [5C modification. Similar to DNA m5C [S-adenosyl-methionine (SAM), is added to the carbon-5 position of the cytosine base in RNA to form the m5C modification, which is likewise a widespread RNA modification detected in messenger RNA (mRNA) and non-coding RNAs including transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), small nuclear RNA (snRNA), micro RNA (miRNA) and enhancer RNA (eRNA) [5C modifications have been reported in many species but its distribution seems to differ. For example, eukaryotic tRNA and mRNA have more m5C modification than bacterial mRNA and tRNA [The discovery of DNA 5-Methylcytosine  . The m5Cand tRNA , 12.5C modifications on tRNA and rRNA were extensively studied. In tRNA, m5C has been shown to maintain homeostasis, optimize codon–anticodon pairing, regulate stress response, and control translation efficiency and accuracy [5C modification is involved in glioma sensitivity to bioactive substrates of the stress-related enzyme NQO1 and structural stability of the tertiary rRNA–tRNA–mRNA complex under stress [5C modification of eukaryotic mRNA, such as hamster cell mRNA and certain viral RNA but not SV40 RNA [5C modifications in mRNA and ncRNA were discovered. To date, mRNA m5C modifications have been associated with various biological processes, such as mRNA stability, splicing and nuclear–cytoplasmic shuttling [5C modifications have been associated with the etiology of multiple diseases, including arteriosclerosis [maccuracy –18. rRNAr stress . Until iSV40 RNA , 21. Lathuttling –24; DNA huttling ; prolifehuttling ; develophuttling , 28. Abeclerosis , autoimmclerosis  and cancclerosis .5C sites in Mus musculus and Arabidopsis thaliana was recently developed [.With the development of high-throughput sequencing technology and the improvement of liquid chromatography sensitivity, the methods for the identification of the overall level of RNA methylation have been greatly developed. The current technical means used for the identification of the overall level of RNA methylation is mainly liquid chromatography coupled with MS (LC-MS). LC-MS/MS uses tandem mass spectrometry based on liquid-phase mass spectrometry, which is able to obtain both molecular and fragment ion peaks, allowing both qualitative and quantitative analysis of bases . In conteveloped .5C modification is another abundant mRNA modification and may be another RNA epigenetic marker [5C modification in LC-MS, the adaptability of bisulfite sequencing with RNA opens a new possibility of mapping m5C with nucleotide resolution in RNA. Using this method, Lukas Trixl and Alexandra Lusser found ~ 10,000 sites showing > 20% methylation and mapping to ~ 8500 mRNAs resulting in a rate of 0.43% m5C of all sequenced Cs, and they also published the first cytosine methylome for human cells [Bisulfite sequencing shows that mc marker . Recentlan cells .5C sites (> 20% methylation) corresponding to 1650 mRNAs in mouse embryonic stem cells and 2075 m5C sites corresponding to 486 mRNAs in mouse brain. Their concluded that m5Cs modification mainly exist in the coding region and are enriched around the translation initiation site [5Cs modification localized to the untranslated regions (UTRs) of mRNA transcripts [Recently, Amort et al. detected about 7500 mion site . Anothernscripts .5C sites in Arabidopsis thaliana mRNA were detected by bisulfite sequencing [5C in the archaebacterium Sulfolobus solfataricus and budding yeast, and a single site in yeast and 14 m5C modified mRNAs in S. solfataricus [Another recently published article on HeLa cells and mouse cytosine, a matrix, identified about 3600 loci in about 2000 genes in HeLa cells and 2500-4400 sites (1000-1655 genes) in different mouse tissues , 39. Aboquencing . MeRIP-sataricus  were dis5C in mRNA is not comprehensive and thorough, while there are some interesting findings, such as Yang et al. demonstrated that during the maternal-to-zygotic transition (MZT) of zebrafish, RNA m5C modification regulates maternal mRNA stabilization, highlighting the key role of m5C mRNA modification in early development [5C pandora-box roles besides in regulating mRNA stability.However, the exploration of the function of melopment . More ef5C not only plays a role in regulating the stability of mRNA, but also regulating the stability of rRNA and tRNA. A large number of studies have reported that m5C modification is vital for translation regulation of tRNA and rRNA [It was found that mand rRNA , 43.5C modification are focused on tRNA. Methylation of tRNA most often occurs on the cytosine at the junction region between the variable ring and the T-stem, as well as on one, two, or three Cs spanning the 47-50 position [Asp can effectively stimulate tRNA charging Asps in vivo and in vitro, hereby promoting the translation of proteins harboring multiple Asps [5C38 of tRNAMet results in a moderated translational fidelity [5C38 tRNAAsp can help to distinguish homologous and near homologous codons, such as different tRNAASP from tRNAglu, avoiding a false positive rate of amino acids, and improve the accuracy of translation [Thr and tRNACys transcription, the CCA sequence is added at the 3’end and thereafter C72 was confirmed to be methylated [5C34 of mt-tRNAmet, and it play a key role in mitochondrial translation as methionine in decoding unconventional AUA codons [5C34 of tRNALeu is associated with translation regulation. In oxidative stress responses, its methylation level is significantly increased, which can enhance the translation of uug-codon-rich mRNAs, such as ribosomal protein El22A [5C48 and m5C49 are located in the junction sequence of the V-loop (VL) and T-stem-loop (TSL), which is essential in accelerating tRNA stability and protein translation , and it prevents Polycomb repressing complex 2 (PRC2) complexes from binding in vitro [5C is distributed in lncRNA and is positively correlated with lncRNA transcription. Studies have confirmed that hm5C directly or indirectly regulates dysregulated colorectal cancer lncRNAs by the abnormal activity of superenhancers and promoters modified by the hm5C. In addition, hm5C also participates in long-range chromatin interactions at the lncRNA sites. They also found that lncRNAs regulated by different hm5C markers were associated with different clinical outcomes and tumor status [It has been reported that if cytosine-5 methylation is absent in the vault RNA, it will be abnormally processed into Argonaute-associated small RNA fragments, which have a similar function as microRNAs . Althougin vitro . Hu et ar status .5C has relatively rich but highly dynamic characteristics, and it constitutes a multifunctional and effective mechanism for coping with constantly changing internal and external environments. It acts by regulating the intracellular RNA metabolism and related functions. m5C modification is increasingly becoming mainstream in the field of epitranscriptomics.As a type of RNA modification, m5C modification is mainly mediated by 3 types of proteins, namely the methyltransferases (writers), the demethylases (erasers) and the binding proteins (readers) respectively. The m5Cs in RNAs are recruited by NOL1/NOP2/SUN (NSUN) protein family, including NSUN1-7 and DNA methyltransferase (DNMT) homologue DNMT2 [mue DNMT2 , NSUN1, ue DNMT2 Fig. 3)..5C modif5C (RCMTs) uses adenosylmethionine as a methyl donor to form m5C by transferring the methyl group to cytosine [5C methyltransferases have been found, including DNMT2, and tRNA-specific methyltransferase (TRDMT) family members, NOL1/NOP2/SUN domain (NSUN) family member. Among them, the NSUN methyltransferases includes several members (NSUN1 to NSUN7) and NSUN5a/b/c [Arabidopsis thaliana [5C modification. Liu and colleagues have solved the structures of NSUN6 [5C methylation.The methyltransferase of mcytosine . RCMTs acytosine . More thUN5a/b/c . The TRDthaliana . Both NSthaliana . DNMT2 athaliana . A quintof NSUN6 . A non-c5C, which may inhibit the effect of 5-methylcytosine on the formation of double-stranded RNA in eukaryotes [1A) along the cytoplasmic tRNAs [5c34 to hm5cm34 (5-hydroxymethyl-20-o-methylcytidine) and f5cm34 (5-formyl-20-o-methylcytidine) along mt-tRNAmet and cytoplasmic tRNALeu [5C metabolism by ALKBH1 have great potential in regulating mitochondrial activity. Moreover, ALKBH1 can also specifically act on a histone dioxygenase-histone H2A [Protein demethylases called erasers, such as the TET family of enzymes, have a reversible effect by mediating the demethylation of written RNA . In recekaryotes . Besideskaryotes . In receic tRNAs . ALKBH1  tRNALeu . Loss oftone H2A .5C binding proteins, such as ALYREF and YBX1, are considered to be a type of barcode reader, which exerts biological effects by recognizing and binding to m5C sites [5C-modified RNA oligos pull-down coupled with mass spectrometry analysis has been applied to a variety of m5C mRNAs, and two reading proteins of m5C mRNA, ALYREF (RNA and export factor binding protein 2) and YBX1, have been identified [5C recognition site of K171. m5C is recognized by the m5C reader ALYREF through the viral RNA transcript installed in the nucleus, which helps them export to the cytoplasm [5C installation in viral transcripts occurred in the nucleus or the cytoplasm, Eckwahl and his colleagues performed RNAs isolation from nucleus and cytoplasm separately, followed by bisulfite treatment and Sanger sequencing; they focused their functional investigations on ALYREF reads on the protein. They found that knocking out the m5C reading protein ALYREF has significant impact on viral protein production and viral replication. The depletion of ALYREF decreased the MLV Gag protein and virus released cells by 5-fold. On the contrary, the overexpression of ALYREF increased the Gag protein level. They also examined whether using the plasmid encoding wild- type ALYREF or the known m5C to recognize the impaired ALYREF mutant (K171A) could rescue the 3 T3 cells lacking endogenous ALYREF. They found that wild- type ALYREF but not mutant constructs can rescue phenotypic effects [5C plays an important role in bridging the interaction between ALYREF and MLV RNA through endogenous RNA immunoprecipitation (RIP). They also confirmed that ALYREF knockout decreased the cytoplasmic and nuclear ratio of m5C in viral RNA, due to fewer m5C containing viral transcripts were exported to the cytoplasm.Most of the biological functions of RNA modification are related to the protein to which it binds. RNA m5C sites . Recentlentified . ALYREF ytoplasm . In orde effects . Eckwahl5C reading protein in human cells [5C in its cold shock domain through the indole ring of W65 [5C-containing oncogenes, such as HDGF, and promotes the stability of these oncogenes and the subsequent progression of cancer by recruiting the well-known mRNA stability maintenance factor ELAVL1.Unlike ALYREF, YBX1 was identified as a cytoplasmic mRNA man cells . Based og of W65 . YBX1 sp5C mRNAs in urothelial bladder cancer (UCB)-derived T24 cells. In zebrafish, the maternal YBX1 loss can increase overall translation, accumulation of unfolded proteins, leading to oogenesis defects and subsequent embryogenesis failure [5C were identified by YBX1. Upon the early development of zebrafish embryos, 397 maternal mRNAs with m5C showed significantly decreased expression when YBX1 was knocked out, indicating that these m5Cs mediated by YBX1 played a key role in regulating maternal gene clearance during the transition from the mother to the zygote [5C reading protein [5C-containing RNAs. Interestingly, like humans and zebrafish YBX1, the highly conserved cold-shock domain (CSD) also contains the YPS m5C binding site. Mutation of this site can induce the defective development of GSC, which indicates that RNA m5C modification has an effect on the development of adult stem cells [Furthermore, YBX1 could recognize about 60% of m failure . Recent e zygote . In Ypsi protein . In Drosem cells .5C modification extensively affects gene expression at multiple levels through interacting with a variety of writer, eraser and reader proteins  database related to HNSCC and performed in-depth sequence analyses. They found that the expression of genes regulated by m5C was associated with the copy number variation (CNV) pattern. Gene set enrichment analysis (GSEA) showed that m5C reader protein ALYREF can actively promote mRNA export, thereby playing the role of an mRNA export adaptor in vivo and in vitro [5C regulatory genes in a variety of tumors with adverse clinical results, and provided a partial explanation for the discussion of cancer pathogenesis and/or survival. He and his colleagues assessed the correlation between the expression of m5C regulatory factors and related cancer pathways, and found that mutations in m5C regulatory genes and CNVs are significantly related in many cancer types. It is worth noting that this study verified the frequency of m5C regulatory patterns in 33 cancers and compared the expression levels of m5C regulatory factors in tumors and adjacent tissues, and found that they were overexpressed in tumors [5C methyltransferase under glioma from the Chinese Glioma Genome Atlas (CGGA) and TCGA data sets. The results showed that RNA:m5C methyltransferase was significantly related to the malignant progression of glioma [Increased levels of RNA mpatients . Increaspatients . Xue et in vitro . Meanwhin tumors . Glioma f glioma .5C regulatory genes were found in HCC. Dysregulation of m5C-related genes was also associated with a higher HCC stage. Moreover, almost all patients with low survival rate were significantly associated with high expression of m5C regulatory factors. High expression of NSUN4 and ALYREF is highly coincident with the survival outcome. GSEA results indicate that methylation and demethylation will lead to high expression of NSUN4 [5C modification of H19 lncRNA mediated by NSUN2 can affect its stability and interaction with the oncoprotein G3BP1, and it has been proved to be related to the poor differentiation of HCC. It indicates that this interaction may serve as a prognostic biomarker for HCC patients [The NSUN2 gene has the highest mutation rate in gastrointestinal cancers . A high of NSUN4 . In addipatients .5C promotes the pathogenesis of human bladder urothelial carcinoma. m5C stabilizes oncogene-mRNAs through the m5C-modified sites in their 3’ UTRs and promotes the pathogenesis of human bladder urothelial carcinoma by [5C methylation in the 3’-untranslated region of HDGF and promote the urothelial carcinoma of the bladder. Moreover, upregulated NUSN2, YBX1, and HDGF expression levels predicted a poorer survival rate in patients with urothelial carcinoma of the bladder [5C in the development of urothelial carcinoma of the human bladder by stabilizing oncogene-mRNAs through m5C modified sites in their 3′ UTRs [A recent study indicated that minoma by . Zhang e bladder . A recen 3′ UTRs .5C and its writer proteins are associated with lineage-specific chromatin structure and treatment response/resistance, and they may be an epigenetic driver of leukemia cytochemical resistance. Studies have shown that NSUN3 and DNMT2 forms 5-azacytidine (5-AZA) sensitive chromatin structure through binding to the RNA binding protein hnRNPK, complexed with GATA1 and SPI1/PU.1, which interacts with CDK9/P-TEFb to recruit RNA polymerase-II in the nascent RNA. However, NSUN1 is recruited to the active chromatin structure formed by BRD4 and RNA polymerase-II. Though this chromatin structure is insensitive to 5-AZA, it is highly sensitive to the BRD4 inhibitors JQ1 and NSUN1 knockdown. Furthermore, RNA m5C and NSUN1−/BRD4 related active chromatin were enriched in 5-AZA-resistant leukemia cells and 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukemia specimens [In addition, it has been shown that mpecimens .5C writer, eraser and reader proteins are found in various human cancers - mediated immune effects of mRNA [5C-mRNA can be used as a new approach in the field of mRNA-based therapeutics, partially due to that m5C-modified mRNA may evade the innate immune system through inhibition of endosomal Toll-like receptor 3(TLR3)  Fig. .Fig. 7Th5C in tumor immune microenvironment. Diverse methylation regulators can be used as prognostic and diagnostic markers of cancer [5C regulators had lower immune activity in TILs such as DCs, NK cells, and CD8+ T-cells [5C-score contained higher resting CD4+ memory T cells and CD8+ T cells and had better prognosis, while activated NK cells and monocytes were mostly enriched in the high m5C-score patients with worse prognosis [5C RNA methylation regulators, the up-regulation of NSUN2 expression and the down-regulation of NSUN6 expression, which can objectively predict the clinical prognostic risk of TNBC patients to a large extent. Therefore, it may become new prognostic markers of TNBC and provide clues for understanding the RNA epigenetic modification of TNBC [5C pattern had different TME immune cell infiltration and high m5C score group had a better prognosis [5C-regulated lncRNAs also predict the overall survival of lung adenocarcinoma patients and affect the tumor immune microenvironment [5C-related lncRNAs showed prognostic value. With TIDE (Tumor Immune Dysfunction and Exclusion) algorithm, high-m5C-lncRNA scores had a better response to immunotherapy [5C-related lncRNAs and PDAC-infiltrating immune cells were evaluated. Naïve B cells, CD8 + T cells, Treg cells, and resting NK cells had a higher expression level in the low-risk group while the M0 and M2 phenotype macrophages had a higher expression level in the high-risk group, suggesting that m5C-related lncRNAs may regulate pancreatic cancer progression via promoting M2 phenotype macrophage polarization or infiltration in PDAC [Increasing evidence based on bioinformatic analysis implicate the significance of mf cancer , 109. Or T-cells . Papillarognosis . In trip of TNBC . Related of TNBC . In lungrognosis . Besidesironment . In pancotherapy . In anot in PDAC . Related in PDAC , hepatoc in PDAC , breast  in PDAC , human h in PDAC , cutaneo in PDAC  and colo in PDAC .5C. NSUN2 is an important factor in maintaining stem cell self-renewal and differentiation [Immune escape is a hallmark of human cancer. T-cell exhaustion mainly accounts for escape of tumors from immune surveillance. NSUN2, a tRNA methyltransferase, modifies tRNA and mRNA by increasing the methylation of mntiation . In headntiation . It has ntiation , thus inntiation . Nonethentiation .+ T cells and downregulation of myeloid-derived suppressor cells and regulatory T cells are associated with the overcome of the tumor immunosuppressive environment and immune escape status. YBX1 drives signal transduction in a tumor immunosuppressive microenvironment and immune escape pathway. In addition, YBX1 knockout can reverse HCC drug resistance by blocking PD-L1 expression and activating T cells in a tumor microenvironment. Data presented by Tao et al. showed that both tumor immune evasion and multidrug resistance can be reversed by targeting the YBX1 signaling cascade, suggesting an effective treatment regimen against tumor chemoresistance [YBX1 protein is the most common tumor-associated antigen and can also induce T cell response . Therefosistance .5C regulators could be a useful strategy.In conclusion, these data implicate that modulation of m5C modifications of RNA have been demonstrated as a new molecular mechanism that controls eukaryotes gene expression. How the modification of m5C affects the ncRNA biogenesis, localization, and function, and how these effects are associated with etiology of cancer are to be elucidated. In recent years, extensive efforts have been devoted to the study of m5C modifications in cancer. In this review, particular attention was focused to Writer-NSUN2, the Eraser-TET family, and Reader-YBX1..m5C modulators targeted by small-molecule inhibitors have therapeutic potential for cancer treatment. As of now, specific m5C inhibitors have not been developed. In DNA methylation, the chemicals initially developed to target these proteins, such as 5-aza-2-deoxycytidine (decitabine) or 5-azacytidine (Vidaza), have been approved to treat myelodysplastic syndrome by the FDA, but their usage in solid tumor treatments is limited.Some proof-of-concept studies have revealed that maladjusted m5C modification will also be critical in both identifying the m5C sites from Single-molecule sequencing and in prediction of m5C sites in different RNA species [5C sites in Homo sapiens, Mus musculus and Arabidopsis thaliana [5C epitranscriptomics will be fully understood and its relation with cancer will be uncovered.Bioinformatic research in RNA m species . Severalthaliana , 132. Wi5C in immune system regulation and tumor immune microenvironment will be important directions. As for RNA m6A, depletion of m6A writers, readers or erasers had significant phenotypic consequences in the cellular response to infection and in a few immune cell types. Whether RNA m5C exerts similar functions in the immune system deserves deep efforts. More importantly, how different RNA modifications combat or combine with each other in immune regulation needs a long way to explore.In the future, the detailed roles of RNA m6A, the RNA m5C modification partially shares the same enzymatic machinery including DNMT1 with DNA m5C. This mechanism may also play critical roles during the tumor development and progression. m5C can operate with the tumor cells and immune microenvironment to brake or accelerate the tumorigenesis. It will be interesting to identify the immune checkpoint molecular target of m5C in the tumor microenvironment, so that targeted therapy can be used in combination with checkpoint immunotherapy to better treat cancer. In general, the investigation of novel m5C epigenetic modification in cancer not only will provide new insights into the molecular mechanisms of cancer biology and immune response, but also will pave the way to the develop new promising therapies.Different from m


The use of immune checkpoint inhibitors (ICIs) as a mono- or adjuvant oncologic treatment is rapidly expanding to most fields of cancer. Alongside their efficacy, ICIs carry the risk of immune-related adverse events (irAEs) arising from misguided immune-mediated response to normal tissues. In the cardiovascular system, the cardiac toxicity of ICIs has been primarily related to the development of an acute, immune-mediated myocarditis; beyond this potentially fatal complication, evidence of an increased risk of cardiovascular events and accelerated atherosclerosis is emerging, as well as reports of other cardiovascular adverse events such as arrythmias, Takotsubo-like syndrome and vascular events. The absence of identified risk factors for cardiotoxic complications, specific monitoring strategies or diagnostic tests, pose challenges to the timely recognition and optimal management of such events. The rising numbers of patients being treated with ICIs make this potential cardiotoxic effect one of paramount importance for further investigation and understanding. This review will discuss the most recent data on different cardiotoxic effects of ICIs treatment. Immune checkpoint inhibitors (ICIs) emerged in the last decade as a rapidly developing field of cancer treatments, and their use is expanding to a wide range of cancer fields . In a siAs is known, PD-L1 is expressed on myocytes, and its signaling path plays an important role in protecting the heart from autoimmune damage . It was Takotsubo syndrome (TTS) is an acute, mostly reversible, left ventricular (LV) systolic dysfunction, characterized by the classic echocardiographic findings of depressed LV functions along with akinetic apex , in the absence of obstructive coronary artery disease. ICIs- associated TTS was reported in several studies and case-reports ,13,28,29Current data regarding ICIs- associated pericardial involvement are limited, but case-reports include pericarditis, pericardial effusion or tamponade ,30,31. TArrythmias, mostly supraventricular tachycardia but also atrial fibrillation, ventricular fibrillation and heart block, have been described in the context of ICIs therapy ,13,28. HImmune checkpoints are established negative regulators of atherosclerosis- and arterial wall disease, which involves a lipid-driven chronic inflammatory process, in which T cells play a dominant role. For example, mice lacking PD-1/PD-L1 pathways demonstrated an increase in atherosclerotic plaque , while CRecent data suggest an increased incidence of myocardial infarction in ICIs trials . Drobni Vasculitis disorders observed in association with ICIs can affect vessels of any size, but were most commonly reported in larger vessels, particularly temporal arteritis . An immuMyocarditis is a well-established life-threatening cardiotoxic adverse event of ICIs therapy. However, with the relative novelty of the ICIs, together with their rapid expansion into all fields of oncologic treatments, short and long-term complications that have not yet been revealed are highly likely . MoreoveThe European Society of Cardiology (ESC) recently published a position statement which recommends a baseline cardiac assessment of all patients scheduled for ICIs treatment initiation, including a clinical history and risk factor assessment, ECG, cardiac troponin, BNP or NT-proBNP and echocardiogram. Having these parameters available at baseline will allow for a better evaluation of patients later on, in case a cardiotoxic adverse event is suspected. In patients categorized as high-risk patients, the current recommended surveillance during ICIs treatment includes ECG, cardiac troponin and BNP/NT-proBNP assessment before ICIs doses 2, 3 and 4; if normal, then further assessment can be reduced. In case of new troponin or BNP elevation, ECG or echocardiographic abnormality, the patient should be referred to a cardio-oncology specialist. Any patient with a new cardiac symptom should undergo prompt evaluation including ECG, echocardiography, cardiac troponin and BNP/NT-proBNP and should be referred to a cardio-oncology specialist if any new abnormalities arise .Given the increasing number of patients treated with ICIs, further research is needed to establish standards of monitoring and best practical management of patients in all potential manifestations of ICIs cardiotoxicities. Once ICIs-related cardiotoxicity is developed, a close collaboration is needed in a multi-disciplinary team in order to outline the most appropriate management and best treatment options in both a cardiac and oncologic sense.


The application of ribo nucleic acids for molecular studies requires high integrity and quality of extracted total RNA samples. In addition, the need to transfer RNA samples at room temperature without special treatments such as ice and liquid nitrogen storage according to international transport laws highlights the importance of low cost alternative methods such as RNA air-drying, lyophilisation and transportable agents. In this study, the quality and quantity of air-dried RNA samples from leaf, petiole and bark tissues of different olive genotypes using several RNA extraction methods were compared with lyophilized ground leaves and RNAlater-stored tissue samples before precipitation. The quality of RNA and prepared libraries were checked by several techniques including agarose and polyacrylamide gel electrophoresis, Agilent quality control, RT-PCR amplification of housekeeping and viral genes and high throughput sequencing.Rbc 1 and Nad 5) and partial genomes of Arabis mosaic virus and Cucumber mosaic virus showed satisfactory results in RNA samples extracted by TRIzol™ Reagents despite its variation amongst cultivars.Although RNA value varied amongst cultivars, RNA extraction with TRIzol™ Reagent in fresh extractions and samples stored in RNAlater before RNA extraction resulted in 455.26 ng/µL and 63.46 ng/µL  as the highest RNA concentration averages, respectively. RNA samples extracted by TRIzol™ Reagents and stored for a short term at – 80 °C before air-drying showed the third highest concentration (44.87 ng/µL). The synthesized cDNAs quality for PCR amplification of housekeeping genes (Considering the difficulties in the extraction of high quality and quantity RNA in olive for molecular analyses, this study demonstrated that RNA extraction method based on TRIzol™ Reagent can be considered for virobiome studies of both fresh and air-dried samples. In comparison with DNA, the physico-chemical stability is more prominent in the case of RNA. Typically, RNA is rapidly degraded and it is vulnerable to hydrolysis by ubiquitous ribonucleases and/or divalent cations. Ribonucleic acids are degraded in several hours or days at room temperature even in the absence of RNase . RNA dethttp://www.iata.org/whatwedo/cargo/dgr/Documents/Acceptance-Checklist-Dry-Ice2014-EN.pdf], there are several limitations for routine transfer of biological materials using dry ice where temperature may still compromise the integrity of the RNA being shipped. RNA molecules are inherently sensitive to a number of factors such as heat, oxidation, pH alterations, and especially cellular RNases [Long-distance shipment of biological samples for international exchange between collaborators or analysis is inevitable . Accordir RNases . Lipid mr RNases  stated tRegarding viral diseases of plants, RNA viruses are still the most prevalent disease-causing agents particularly in deciduous plants . Virus dTortula ruralis (Hedw.) [In the lyophilisation technique, RNA solvent, which is diethyl pyrocarbonate (DEPC) treated deionized water, is typically removed from frozen sample via sublimation . Lyophil (Hedw.) .Several limitations such as low antigen titre specifically in phloem restricted viruses , 12, irrIn the present study, some total RNA extraction methods including two modified TRIzol- and CTAB-based methods and two commercial RNA extraction kits  were used for RNA extraction from different olive genotypes. The results were then compared to RNAs derived from lyophilized tissues and RNAlater immersion samples which later precipitated with Spectrum plant total RNA kit to obtain the highest quality and integrity RNA.The results of RNA extraction and contamination in two different tissue types are shown in Table Rbc 1 and Nad 5), and of Arabis mosaic virus (ArMV), and Cucumber mosaic virus (CMV) genomes from several cultivars are also shown. All the RNAs extracted manually or by commercial kits showed acceptable quality on gel electrophoresis except for modified CTAB 2 protocol. The housekeeping genes were amplified successfully using related synthesized cDNAs  and Piwi-interacting RNA (piRNA) bands normally range between 140 and 150 bp, respectively while adaptors are ligated  on 1.5% agarose gel. Ethidium bromide staining of agarose gel electrophoresis showed two sharp bands corresponding to 18 S and 28 S rRNA for samples in fresh extractions  showed different concentrations Table . TRIzol For fresh methods they were measured immediately after extraction, in case of transportable methods they were measured after 10 days of air-drying, and in case of complementary methods they were measured after storing at room temperature for 10 days and later were used for RNA extraction (Mean  ±  SE).A260/280 ratio values of RNA ranging from 2.03 ± 0.27 to 2.10 ± 0.04 in fresh and air-dried RNAs obtained from CTAB 1, which can be considered as the least contaminated in this study .The FastQC results of sequenced metavirome of air-dried RNA samples extracted by TRIzol 1 showed that quality score, known as Phred or Q score, across all bases is an integer value representing the estimated probability of an error showing the base is incorrect. Phred score of 20 or above is acceptable; however in the present study Q score was around 36 except for three starter bases which normally were trimmed during bioinformatics analysis. In this case, an incorrect base call was less than 1 in 1000 meaning more than 99.9% confidence  which were lyophilized and stored at room temperature for 0, 3 and 6 weeks [RNA isolation from plant tissues is generally more challenging than animal tissues. The rRNAs and tRNAs are two types of RNA numerously found in all cells and are mostly extracted in the same proportion . It is r 6 weeks . Several 6 weeks . Lyoprot 6 weeks .However, lipidoid nanoparticles (LNPs) were able to be lyophilized without diminishing their potency , furtherIn the present study, TRIzol 1 can be considered a suitable method for overseas exchanges and sequencing as it is reported to be efficient specifically in small RNA studies . NoteworCTAB as a well-known methodology is applicable for a wide range of tissues and species such as apple, pear, potato, thyme, wheat, rice, etc. The accuracy of its results for PCR, RT-PCR, real time-PCR and southern blot analysis have been proved . In thisAbnormal 260/280 ratios usually indicate that a sample is contaminated by residual phenol, protein and guanidine . These tV. vinifera cv. Chardonnay) on the gel electrophoresis [The RINs greater than 7 are generally considered excellent for use in RNA-Seq application . The resphoresis . Read maphoresis .Since the application of virus-free planting materials is of great importance in sustainable agriculture, the need to accurately detect plant materials infected with viruses has an applicable significance. Olive genotypes as valuable horticultural products host several viruses from different genera. Molecular biology studies regarding olive total RNA and also phytopathological investigation necessitate RNA stabilization for a longer period.RNA integrity and value is highly cultivar dependent. For fresh extraction studies, TRIzol reagent performance in olive cultivars RNA mean value was prominent. In shipment cases, despite RNA stabilizer superiority in RNA value, air-dried cases extracted with TRIzol reagent were more satisfactory in agarose gel electrophoresis, cDNA synthesis, library preparationn, and RT-PCR amplification of both housekeeping and virus genes. Although secondary metabolites vary significantly amongst olive cultivars, RNA extraction methods are prominently determinative in contamination co-purification with targeted RNA. CTAB 1 showed an acceptable range of contamination (A260/280 and A260/230), which is highly attributed to its chemistry. Consequently, RNA air-drying can be considered a promising method in the out-of-ice shipment process of plant materials, where there is a scope for improvement.Samples of leaves, petioles and bark tissues with three replications were collected randomly from actively growing shoots of 70 mature olive trees with ascertained genetic trueness from Olive Research Station  in June 2019, possessing the main sources for olive improvement in Iran . We statAll glassware and tips used for RNA extraction were autoclaved twice for 45 min  unless otherwise stated, and all pliers were oven dried at 200 °C overnight. All chemicals were of analytical grade. To optimize RNA concentration in different tissues, leaf RNA and stem bark of ‘Conservolia’ cultivar were compared and a combination of leaf, petiole and bark tissues were selected for all other cultivars.Total RNA from each sample was extracted using TRIzol™ Reagent . One millilitre of TRIzol™ was added to 100 mg of homogenized sample, mixed well and centrifuged. The supernatant was mixed with 200 µL chloroform  and recentrifuged. The resultant supernatant was mixed with 250 µL isopropanol , and 250 µL NaCl (2 M)  and centrifuged again. Pellet was washed twice with 75% (v/v) ethanol , resuspended in 25 µL DEPC  treated deionized water, incubated at 65 °C for 15 min and stored at − 80 °C. All centrifugations were done for 15 min at 13,000 rpm and 4 °C. In TRIzol 1, RNAs were stored at − 80 °C for a short time before air-drying, whereas in TRIzol 2 samples were immediately air-dried.This protocol was done according to Doyle and Doyle  with somThis method was done according to Zarei et al.  using twFor RNA extraction, 1 mL pre-heated buffer I was added to 100 mg of fine ground plant tissues of cultivars ‘Amin’, ‘Meshkat’, ‘Tokhm-e-Kabki’, ‘Dire’, ‘Arbequina’, ‘Conservolia’, ‘Koroneiki’ and followed by adding 20 µL β-mercaptoethanol. Tubes were thoroughly vortexed and incubated  and centrifuged . The supernatant was transferred to new tube and equal volume of chloroform:IAA (24:1) was added, vortexed and centrifuged  and this step was repeated. Next, ¼ volume of aqueous upper phase, LiCl (1 M) was added to the resultant supernatant and was gently inverted and incubated at 4 °C overnight. The sample centrifuged for 40 min at 12,000 rpm and 4 °C. This supernatant was decanted and tubes were gently tapped on autoclaved tissue paper . The pellet was washed with 500 µL ice cold 70% ethanol and centrifuged shortly. In the next step, 300 µL pre-heated buffer II was added to dissolve the pellet. An equal volume of 24:1 chloroform:IAA was added and centrifuged for 10 min at 10,000 rpm and 4 °C. Then, 0.1 volume of sodium acetate (NaOAc)   and 0.6 volume of isopropanol were added, mixed and incubated at − 20 °C for at least 2 h. Tubes were centrifuged for 20 min at 12,000 rpm and 4 °C and the pellets were washed with ice cold 70% ethanol and dried for 15 min on tissue paper. The pellets were resuspended in 30 µL PEPC treated deionized water.Two commercial RNA extraction kits namely Ribospin Plant RNA Extraction Kit  and Spectrum Plant Total RNA Kit  were used, according to respected manufacturer’s illustrations.The extracted RNAs of all cultivars were first adjusted to the same quantity and quality (A260/A280). The RNAs extracted with TRIzol 1, 2, CTAB 1 methods and Ribospin Plant RNA extraction kit, were precipitated with 0.1 volume sodium acetate (3 M) and 5 volume ice cold 100% ethanol. Samples were vortexed thoroughly and kept at − 20 °C overnight. Then, the samples were centrifuged for 30 min at 13,000 rpm and 4 °C. The RNA pellets were washed with 0.5 mL ice cold 75% (v/v) ethanol, span for 10 min and stored at room temperature for 10 days. Air-dried total RNAs were shipped out–of-ice and diluted in respected buffer from Spectrum ™ plant total RNA kit  after 10 days for further analyses.Samples of leaves, petioles and bark tissues of each cultivar  were first ground in liquid nitrogen to fine powder and then lyophilized for 18 h at − 60 °C by vacuum freeze dryer . Lyophilized samples were stored at room temperature for 10 days before RNA extraction. RNAs were extracted by Spectrum ™ plant total RNA kit  according to the manufacturer’s instruction.Samples  consisting of leaves, petioles and bark tissues were immersed in RNA stabilizer solution  and stored at room temperature for 10 days before RNA extraction by Spectrum plant total RNA method .www.caliperLS.com, Application Note 402).The quality and integrity of extracted RNAs were assessed by several methods including spectrophotometry for A260/280 and A260/230, appearance on a denaturing 1.5% agarose gel, ability to amplify RT-PCR products and fluorimetry  just after elution in both fresh extracted and lyophilized samples (before storage at − 80 °C). The RNA Integrity Number (RIN) were tested by Agilent 2100  referring to the amount of different RNAs. An algorithm is generated by taking multiple samples and manually assigning them all a value of 1–10 based on their integrity which 10 represent the least degradation. RQS vs RIN (with 10% CV) is calculated byRubisco  with primer pair F-TACTTGAACGCTACTGCAG and R-CTGCATGCATTGCACGGTG [Nad 5 , and two olive viruses including Arabis mosaic virus (ArMV), and Cucumber mosaic virus (CMV) using the previously reported primer paires [® Sequencing clean-up kit .RNA of all cultivars was subjected to cDNA synthesis using random hexamer primer and M-MuLV reverse transcriptase  according to manufacturer’s instruction for fresh and transferred samples. The cDNA was tested for several housekeeping genes including GCACGGTG  and Nad r paires , 34 as q® Ultra™ II multiplex small RNA Library Prep set  according to the manufacturer’s instructions for MiniSeq Illumina® . Regarding Agilent assessment, gel-dye mix was allowed to equilibrate to room temperature for 30 min before use. Next, a new High Sensitivivity DNA chip was put on the chip priming station. Then 9 µL of gel-dye mix was pipetted in the well marked as G. The plunger was positioned at 1 mL and then the chip priming station was closed. Plunger was pressed until it was held by the clip. It was waited for exactly 60 s then clip was released. Next it was waited for 5 s, then the plunger was slowly pulled back to the 1 mL position. The chip priming station was opened and 9 µL of gel-dye mix was pipetted in the wells marked G. Next, 5 µL of marker (green) was pipetted in all sample and ladder wells. One microliter of High Sensitivity DNA ladder (yellow) was pipetted in the well marked as ‘ladder’. Then, 1 µL of sample and marker was pipetted to used wells and unused wells, respectively. Finally, the chip was put horizontally in the adapter, vortexed for 1 min at 2400 rpm, and was run in the Agilent 2100 Bioanalyzer instrument within 5 min.The cDNA libraries were prepared using NEBNextP  ≤ 0.01.The RNA extraction experiments were conducted under a completely randomized design with at least 3 replications. Data were evaluated by the analysis of variance (ANOVA) according to the general linear model (GLM) procedure using statistical software SPSS Statistics 22  and mean comparisons among treatments were conducted using Duncan test at the


Influenza A virus (IAV) is a member of the single-stranded RNA (ssRNA) family of viruses. The most recent global pandemic caused by the SARS-CoV-2 virus has shown the major threat that RNA viruses can pose to humanity. In comparison, influenza has an even higher pandemic potential as a result of its high rate of mutations within its relatively short (<13 kbp) genome, as well as its capability to undergo genetic reassortment. In light of this threat, and the fact that RNA structure is connected to a broad range of known biological functions, deeper investigation of viral RNA (vRNA) structures is of high interest. Here, for the first time, we propose a secondary structure for segment 8 vRNA (vRNA8) of A/California/04/2009 (H1N1) formed in the presence of cellular and viral components. This structure shows similarities with prior in vitro experiments. Additionally, we determined the location of several well-defined, conserved structural motifs of vRNA8 within IAV strains with possible functionality. These RNA motifs appear to fold independently of regional nucleoprotein (NP)-binding affinity, but a low or uneven distribution of NP in each motif region is noted. This research also highlights several accessible sites for oligonucleotide tools and small molecules in vRNA8 in a cellular environment that might be a target for influenza A virus inhibition on the RNA level. Orthomyxoviridae family of viruses , respectively. They targeted the following regions: 398–411, 181–194, 63–73, 160–174, and 861–874 nt, respectively. In the context of the availability of vRNA8 complementary regions to these oligonucleotides in the cellular environment, the inhibition results are mostly in agreement with the determined herein secondary structure of vRNA8. The target regions fold the same in local and global vRNA8 structure prediction, except at the target region for 867-14L. The 867-14L binding site base-pairing probability is low in MFE  ,62. Notapanhandle structures appeared to be an efficient and specific anti-influenza inhibitor [Influenza virus and its fast evolution brings us to the point that the most effective and universal antiviral strategy should be based on the most conserved RNA structural elements. The aforementioned strategy’s success relies on accessibility of the target regions for base pairing. Effective inhibitors targeting double-stranded RNA (dsRNA) regions also depend on knowledge about RNA structure and accessibility. An example might be peptide nucleic acids (PNAs) which can form a triple helix with targeted dsRNAs . Modifienhibitor .Small molecules (SMs) targeting RNA are another promising antiviral approach . Specifi2 environment at 37 °C.All experiments concerning the virus were performed in Madin Derby Canine Kidney (MDCK) cell line . Cell cultures were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). The culture was incubated in a 5% CO6 per 10 cm2 and grown to reach 100% confluency. Next, the cells were overlaid with the A/California/07/2009 (H1N1) viral dilution in the infection medium  at 0.01 MOI (multiplicity of infection). After an hour, the viral dilution was aspirated, and the post-infection medium -treated trypsin, completed with DMEM) was added and cells were kept at 33 °C, 5% CO2. After 48 h, the viral inoculum was harvested, aliquoted, and kept frozen at −80 °C before titration. Virus titration was performed with standard IFA (Immunofluorescence Focus Assay) [Before infection, MDCK cells were seeded at a density of 3 × 10s Assay) . For theThe infection of the MDCK cells was carried out as described above, with the difference being that the cells were incubated in the post-infection medium for 24 h before the lysis. Next, the cells were washed twice with 1xPBS and incubated with 6 mL of cell lysis buffer prepared according to a published protocol . After tTemplates for the in vitro transcription were PCR-amplified from the pUC19 plasmid, which contained the complete vRNA8 sequence, constructed previously . The PCRFor the in vitro transcription of vRNA8, 1 µg of template PCR product was used. The reaction was performed according to the manufacturer’s protocol . The RNA was cleaned on an RNeasy MinElute column  and checked for integrity on a 1xTBE 1% in the presence of RNA marker .All the oligonucleotides used in this study were synthesized in the MerMade12 synthesizer according to established procedures ,73. AfteCell lysates were thawed on ice and 180 µL of lysate supplemented with RNase Inhibitor (5U) were used per reaction. Briefly, 5 pmol of in vitro transcribed vRNA8 was heated for 5 min at 65 °C, cooled to 37 °C, and immediately added to the lysate for 30 min incubation at 37 °C. For the probing reaction, two types of chemical reagents were chosen—DMS  and 1M7 (SHAPE), the concentrations of which were determined empirically. The 1M7 was synthesized according to published protocols . The cheThe concentration of RNA was measured  and separated in 1%/1xTBE agarose gel electrophoresis. After the confirmation of RNA quality, the reverse transcription (RT) reaction was performed. RT was conducted with SuperScript IV  according to the manufacturer’s protocol with some modifications. Briefly, the 30 µL of the final reaction was combined with 16.5 µL of RNA, 1xFS buffer, 0.5 mM dNTP , and 2 µM of fluorescent-labeled primer   and incuDNA sequencing ladders were synthesized according to the manufacturer’s protocol with modifications . Briefly, for the sequencing reaction, 90 ng of a template (PCR product) was mixed with 5 pmol of labeled primer, one of the 2′-3′-dideoxy-NTP , 0.5x reaction buffer, and 2U of Thermo Sequenase DNA polymerase. The reaction was PCR-amplified and ethanol-precipitated.®EvaGreen® qPCR Mix (no ROX) . All the samples were set up in triplicate, and negative control of total RNA from mock-infected MDCK cells was used.As a reference standard for the quantitative PCR the vRNA were used. After vRNA in vitro transcription from pUC19 plasmids, the standards were reverse transcribed using SuperScript III according to the manufacturer’s protocol, and serial 10-fold dilutions were prepared. The primers matching short fragments of chosen vRNAs , cells were lysed with lysis buffer . The total protein in cellular lysates was stained with Coomassie Brilliant Blue R-250 Staining Solution . Additionally, IAV proteins expression was detected by Western blot analysis using rabbit anti-IAV NP  and mouse anti-IAV HA  antibodies with HRP-coupled anti-rabbit  and anti-mouse secondary  antibodies, respectively. Expression of actin, detected with mouse anti-actin monoclonal antibody (ACTN05(04), Invitrogen) and anti-mouse HRP-conjugated antibodies , served as a loading control .The results of capillary electrophoresis in ABI format were analyzed with ShapeFinder software . To overpanhandle) was introduced. For the local folding prediction, a maximum pairing distance was implemented at 150 nt, and the promoter structure region was excluded from the structure folding.The global secondary structure prediction of vRNA8 was generated using RNAstructure Version 6.2 ,82,83. AThe predicted structures were converted to dot-bracket files in RNAstructure and visualized in PseudoViewer2.5 . For theAll available vRNA8 sequences were obtained from the NCBI Influenza Virus Database. The selection criteria were nucleotide sequence, influenza A virus type, segment 8 (MP), full-length only. This resulted in 34,248 IAV sequences. The coding RNAs were then reverse transcribed using Biopython’s reverse complement function and aligned using MAFFT (FFT-NS-1 method). Custom scripts were then used to map and align vRNA8 motifs to these alignments as well as count the occurrences of nucleotides at each base pairing location. Conservation of base pairing was calculated to give the percent conservation of canonical base pairing , as well as a measure of inconsistent, or potentially non-canonical, pairing. Although rare in full-sequence databases, ambiguous nucleotides (such as N for any) were considered non-canonical in conservation calculations. The inclusion of these base pairings may result in slightly higher calculated levels of conservation but not in a way that would be considered statistically relevant.iS is the Shannon entropy for nucleotide i, i,jP is the probability of base pairing for nucleotides i and j, and N is the sequence length. Probabilities are calculated using the partition function in RNAstructure [The per nucleotide Shannon entropy was calculated using the following:tructure , incorpotructure ,87.like system to gain valuable information that further could also be used for better interpretation of in-cell data. Here, we used such in vivo-like cellular environment conditions for vRNA of IAV.Information about vRNA secondary structure in the cellular environment is highly anticipated for better understanding of influenza biology and designing therapies. At the same time, challenges in conducting and interpretation of in-cellular RNA structural probing create a need for a simpler, in vivo-The secondary structure of vRNA8 of the A/California/04/2009 (H1N1) strain folded in the presence of the cellular and viral components was analyzed and predicted based on chemical mapping data. A multipronged folding approach allowed us to highlight important and highly probable motifs. vRNA8 prediction in RNAstructure and experimental data with and without pairing distance restriction showed different folding when compared to the in vitro structure. Interestingly, the highest calculated probabilities for single- and double-stranded regions of vRNA8 structures in cell lysate were obtained in seven motifs predicted also in vitro for the same strain. A total of 12 of 17 predicted hairpin motifs were present in both global and local approaches with base-pairing distance restrictions. The comparison of Shannon entropy and SHAPE reactivity allowed us to distinguish seven well-defined motifs: 261–288, 312–327, 374–403, 553–589, 677–687, 698–713, and 792–814 nt. Three motifs, 216–260 nt, 261–287 nt, and 312–327 nt, were predicted before for the distant IAV strain A/Vietnam/1203/2004, while the motifs 312–327 nt, 500–547 nt, 645–666 nt were predicted in silico. The motifs 261–288, 312–327, and 797–814 nt are also found for vRNA8 in virio and ex virio of various IAV strains. Importantly, the conducted study also indicated accessible regions of vRNA8 in cellular environment. The previous research showed that the best antisense oligonucleotides targeted, confirmed herein, unstructured and accessible vRNA8 regions: 398–411, 181–194, 63–73 and 160–174 nt.Structure conservation calculations for predicted vRNA8 motifs in a cellular environment revealed high levels of conservation across many IAV strains. The conserved RNA motifs appear to fold independently of regional nucleoprotein-binding affinity, but a low or uneven distribution of nucleoprotein (NP) in each motif region is noted. Some of the motifs were predicted exclusively in either global or local predicted structures. That indicated the importance of considering multiple approaches during the structure prediction, as some important structural features might be omitted by limiting the prediction to only one folding method. The presented research reveals conserved motifs that may play a crucial role during the viral replication cycle. The vRNA8 conserved motifs and knowledge of vRNA8 accessibility could be used for universal anti-viral therapy based on RNA structure.


The study shows that higher RIN values were obtained for non-diseased breast tissue up to 190 minutes after resection and prior to stabilization. Further experimental exposure up to 180 minutes had no significant effect on RIN values. This study strengthens the rationale for assessing RIN and specific gene transcript levels as an objective method for determining how suitable RNA will be for a specific research purpose (“fit-for purpose”).High quality human tissue is essential for molecular research, but pre-analytical conditions encountered during tissue collection could degrade tissue RNA. We evaluated how prolonged exposure of non-diseased breast tissue to ambient room temperature (22±1°C) impacted RNA quality. Breast tissue received between 70 to 190 minutes after excision was immediately flash frozen (FF) or embedded in Optimal Cutting Temperature (OCT) compound upon receipt (T0). Additional breast tissue pieces were further exposed to increments of 60 (T1 = T0+60 mins), 120 (T2 = T0+120 mins) and 180 (T3 = T0+180 mins) minutes of ambient room temperature before processing into FF and OCT. Total exposure, T3 (T0+180 mins) ranged from 250 minutes to 370 minutes. All samples (FF and OCT) were stored at -80°C before RNA isolation. The RNA quality assessment based on RNA Integrity Number (RIN) showed RINs for both FF and OCT samples were within the generally acceptable range (mean 7.88±0.90 to 8.52±0.66). No significant difference was observed when RIN at T0 was compared to RIN at T1, T2 and T3 , or when RIN was compared between T1, T2 and T3. RNA quality assessed by quantitative real-time PCR (qRT-PCR) analysis of  Biobanks contribute to the success of translational research by managing the procurement, processing, annotation, storage, and distribution of biospecimens. These biospecimens, together with their associated clinical data, are critical to our understanding of disease mechanisms and the discovery of new biomarkers. How a sample is handled and processed could affect the quality of RNA extracted from it. RNA degradation could occur due to a number of factors including but not limited to time between surgical resection and stabilization or maintaining tissue samples in suboptimal temperature conditions before stabilization. Such pre-analytical conditions could impact tissue quality and subsequently the RNA derived from these tissue samples.The time between surgical resection and delivery of a tissue sample to a biobank for stabilization and storage will be collection site dependent. Delivery time could range from less than thirty minutes to a couple of hours depending on the resources available at the collection site to support research. Since biobanks are focused on maximizing the use of available tissue and ensuring that tissue available for research is of the quality needed to perform the analysis they are requested for (“fit-for-purpose”), it becomes important to understand how some pre-analytical conditions, for example, time between receiving and stabilization impact tissue quality.Using RNA of poor or compromised quality for experiments could generate unreliable or irreproducible data. Currently, RNA quality is commonly assessed by RNA Integrity Number (RIN) using the Agilent 2100 Bioanalyzer technology with a RIN output of 1–10, where 1 represents the most degraded RNA profile and 10 the most intact .t) of each target RNA can be measured, and because the Ct value represents the number of cycles required for the fluorescent signal from an amplified template to cross the background threshold, it is inversely proportional to the starting amount of intact target RNA. Greater amounts of starting RNA (or intact/non- degraded RNA) will display earlier threshold crossings, that is, lower Ct values. The Ct value is a useful determinant of specific RNA integrity and an objective measure of the concentration of intact target RNA. As a “rule of thumb”, Ct values of ≤29 indicates the presence of abundant and intact target nucleic acid in the sample, values of 30–37 represent moderate intact target nucleic acid, while values of 38–40 represent minimal intact target nucleic acid [Although RIN is an objective method for estimating the quality of RNA, it cannot unequivocally predict the integrity of a specific RNA transcript in a sample. Thus, despite the wide use of RIN as a quality control parameter, there is a need to determine the extent to which it can be used as the basis to accept or reject research samples. This is important because not all studies target the same RNA transcripts, and samples with RIN values that do not work for one experiment may be useful for a different experiment. The use of another independent method to accurately establish RNA quality in addition to RIN values may be an option in research and biobanking. A method that can determine the integrity of specific RNA transcripts is quantitative real-time PCR (qRT-PCR). In qRT-PCR, the threshold cycle  was approved by Advarra Institutional Review Board.Breast tissue obtained from right and left breast of three female donors was transported from the surgery suite to the pathology laboratory for clinical diagnosis and from the pathology laboratory to the biobank for stabilization and storage. At the biobank, each breast tissue was trimmed of excess fat and sectioned into multiple pieces. Representative tissue from each donor was then stabilized in two ways to simulate the routine processing and preservation procedures at our biobank: 1) by flash freezing (FF) in liquid nitrogen, and 2) by embedding in OCT prior to storage in -80°C freezers. The time interval from surgical removal of the three mammoplasty cases, to depositing at the biobank is represented by T0, the shortest time to stabilization for each sample. T0 ranged from 70 to 190 minutes and represents the tissue associated condition or pre-analytical/pre-experimental condition. This condition which is outside the biobank’s control could potentially impact tissue quality. Additional processing by FF and OCT embedding for the remaining donor tissue was performed after exposure to room temperature , 2 hours (T2 samples) and 3 hours (T3 samples). Thus, the total time from surgery to stabilization for the breast tissue samples at T1 to T3 ranged from 250 to 370 minutes. All samples (T0 to T3) were prepared in duplicate and stored at -80°C until RNA isolation. This information is summarized in 260/280) were determined for each RNA sample using the Nanodrop 1000 . All RNA samples were stored at -80°C until RIN assessment and qRT-PCR.Total RNA was isolated from 90-120mg of each FF sample using the RNeasy Lipid Mini Kit  following the manufacturer’s protocol. For each OCT sample, RNA was isolated from 5–10 sections (20μm thickness) with a total weight of approximately 400mg. The OCT sections were transferred into five microcentrifuge tubes and RNA isolation carried out with the RNeasy Lipid Tissue Mini Kit  following the manufacturer’s instructions. RNA concentration (ng/μL) and purity (Abeta-actin (ACTB), glyceraldehyde-3-phoshate dehydrogenase (GAPDH), cyclophilin A (CYPA) and porphobilinogen deaminase (PBGD) using the forward and reverse primers presented in The quality or integrity of the RNA isolated from the samples was determined by measuring the RIN and quantifying the level of four RNA transcripts by qRT-PCR. The RIN was determined with the 2100 Bioanalyzer using the RNA 6000 Nano Kit . Quantitative Real-Time PCR was performed with the iCycler iQ Real-Time PCR Detection System using the iQ SYBR Green Supermix Kit . The qRT-PCR targeted the RNA transcripts for the following: To synthesize the cDNA, 1μg of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcriptase (RT) Kit per the manufacturer’s protocol  using a final reaction volume of 20μL. The final qRT-PCR reaction volume was 50μL, consisting of 2μL of cDNA sample, 1μL of each primer mix and 25μL of iQ SYBR Green Supermix and 22μL nuclease free water. A 40-cycle protocol was run for all of the reactions as follows: denaturation for 1 minute at 95°C, primer annealing for 1 minute at 55–68°C and extension for 1 minute at 72°C. All samples were run in duplicate, and No Amplification (NAC) and No Template (NTC) Controls were included.t values at the different time points to determine how extended exposure to room temperature affected the level of the RNA transcripts of ACTB, GAPDH, CYPA and PBGD and the impact of RIN on Ct values. Based on the experimental design, data were compared using either paired t-test or paired Wilcoxon signed rank test depending on the normality of the data. Data were further analyzed using a mixed model of random effects and fixed effects. If the assumption of normality for the mixed model was not met, and the log transformation of the data was normal, then log-normal data were used for the mixed model analysis. All results were considered statistically significant when p<0.05 using a two-tailed test.Yield, purity, and RIN of RNA from the extended exposure time points were compared to the baseline of each breast sample. Also compared were C260/280 absorbance ratio), mean values were between 2.08 and 2.12 for the FF samples and between 2.0 and 2.08 for the OCT samples indicating high purity or limited contamination with genomic DNA or proteases [Mean RIN values (± standard deviation) for the samples were between 7.88±0.90 and 8.25±0.65 for the FF and between 8.10±0.33 and 8.52±0.66 for the OCT samples . OCT samRNA quality at T0, which is the status of the tissue at receipt, was not significantly different from the RNA obtained after an additional 60 minutes of exposure at ambient temperature (T0 vs T1) for FF (p = 0.43) and OCT . This observation is opposite to the expectation since RNA degradation would be expected from prolonged room temperature exposure, and this would be associated with increase in Ct values. However, Ct values for OCT samples were not significantly affected by the length of time the samples were exposed at room temperature [CYPA (p = 0.177), GAPDH (p = 0.407), PBGD (p = 0.521) and ACTB (p = 0.102); Mixed model of random and fixed effects was performed to determine the effect of extended exposure to room temperature and RIN on Ct with RIN, for the FF samples, we observed that when RIN increased, Ct values significantly decreased for CYPA (p<0.0001), GAPDH (p = 0.0009), PBGD (p = 0.009) and ACTB (p = 0.022) (CYPA (p = 0.177), GAPDH (p = 0.407), PBGD (p = 0.521) and ACTB (p = 0.102); t values significantly decrease with increasing RIN for ACTB (p = 0.007), GAPDH (p = 0.004), and CYPA (p = 0.0002) except for PBGD , CYPA (p = 0.0004), PBGD (p = 0.013), with a trend toward significance for GAPDH (p = 0.056) (For the association of C= 0.022) . Althoug= 0.056) .In this study, we determined how RNA quality from non-diseased breast tissue (reductive mammoplasty) is impacted by prolonged exposure to ambient room temperature 22°C±1°C) before flash freezing or embedding in OCT. It is important to note that T0  represents the time between tissue excision to stabilization at the biobank and the shortest T0 was 70 minutes while the longest T0 was 190 minutes . The bio°C beforeNo significant difference was observed when RIN at T0 (time from resection to delivery at the biobank) was compared to T1, T2 and T3  indicatiACTB, GAPDH, CYPA and PBGD. We observed the lowest Ct value of 14.1 for ACTB (FF) and the highest Ct value of 24.9 for PBGD (OCT). The Ct value represents the number of replication cycles required to produce fluorescent signal above the threshold, and the lower the Ct values, the more abundant the transcript at the start of the analysis. Threshold values of ≤29 indicate the presence of abundant intact target nucleic acid in a sample [t values, we conclude that the gene expression levels of the housekeeping genes were high when samples were received at the biobank and remained high during the additional experimental exposure to room temperature before stabilization by FF and OCT embedding.Although the RIN values obtained in our study were indicative of relatively good quality RNA, it was important to determine if specific RNA transcripts were intact in the FF and OCT samples. Using qRT-PCR, we investigated transcripts of t value for ACTB, GAPDH, CYPA, and PBGD in the FF samples; for the OCT samples, this relationship was not significant, but the same trend was observed except for PBGD  and OCT (23.8 to 25.3) samples compared to those for ACTB, GAPDH, and CYPA which were below 21  to time at receipt (T0) and studies have reported changes that have occurred less than 20 minutes after resection (20). Our study also focused on normal breast tissues, and our observations may or may not be extrapolated to diseased breast tissue. Studies have shown tumor samples, for example, from colorectal cancer to exhibit higher but less variable RIN values compared to normal samples less than 20 minutes after resection . Also, ot ranges 14.1 to 25.3). The results presented here may guide the biobank in its sample processing workflow design including opportunity to triage samples for processing as they are received based on knowledge of the time between resection and receipt at the biobank.In conclusion, using normal breast tissue received in our biobank from a local medical center with a receiving time of 70–190 minutes after sample resection, which reflects a real-life sample receiving experience, we show that the RNA quality determined by RIN and RT-PCR of four housekeeping genes was high at receipt and after 1–3 hours of additional room temperature exposure  and Optimal Cutting Temperature (OCT) processed samples at different time points which include—RIN values, concentration (ng/ul), yield (ug) and 260/280 ratios and sample C(XLSX)Click here for additional data file.


Parkinson’s disease (PD) is a widespread neuronal degenerative disorder with unexplored etiology. It is associated with various pathological events. In particular, the prefrontal cortex Brodmann area 9 (BA9) region is affected in PD. This frontal lobe brain region plays an important role in cognitive, motor, and memory-related functions. BA9 develops Lewy bodies in PD patients and shows essential changes in transcriptome and proteome, connected with mitochondria related pathways, protein folding pathways, and metallothioneins. Recently, altered adenosine to inosine mRNA editing patterns have been detected in various neurological pathologies. In this article, we present an investigation of differences in A-to-I RNA editing levels and specificity of mRNA editing sites in brain tissues of healthy and PD patients based on RNA sequencing data. Overall, decreased editing levels in the brains of PD patients were observed, potential editing sites with altered editing during PD were identified, and the role of different adenosine deaminases in this process was analyzed. Parkinson’s disease (PD) is the most common age-related motoric neurodegenerative disease that greatly affects one’s overall quality of life. The etiopathogenic mechanisms that underlie this illness are not yet fully understood. PD is considered to be a disease of the neuronal system characterized by the depletion of dopamine neurons in substantia nigra pars compacta  and the Besides the dopaminergic nigrostriatal system, PD also affects the frontal and prefrontal cortex ,5. Thus,To date, a large number of studies have shown that adenosine to inosine (A-to-I) mRNA editing is involved in neurological and neurodegenerative diseases in humans . A-to-I https://www.ncbi.nlm.nih.gov/bioproject/PRJNA283498, accessed on 7 November 2020). For RNA extraction, brain samples were homogenized in TRIzol , then total RNA fraction was isolated using the Qiagen RNeasy Mini Kit  and purified using Agencourt RNA clean magnetic beads . RNA-Seq library preparations were made using Illumina’s TruSeq RNA Sample Prep Kit according to the manufacturer’s protocol. Samples were sequenced using 2 × 100 nt paired-end runs on Illumina’s HiSeq 2000 system [Raw sequencing reads of post-mortem prefrontal cortex (BA9) samples from 29 individuals with PD and 44 neurologically healthy individuals were analyzed . All con0 system . http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 7 November 2020). The quality assessment of the sequences was performed using FastQC software v0.11.8  to a minimum length of 25 nt using Trimmomatic v. 0.39 . TrimmedThe REDItools (v 1.2.1) software was used (REDItoolDenovo.py) to identify potential RNA editing sites in RNA-Seq data. We tuned a list of REDItools parameters to identify more potential editing sites, multi-hits and duplicates from reads alignment were excluded , and only reads in concordant pairs were used (parameter -p). The filter for mapping the quality score was set to 60 (parameter -m) according to REDItools recommendations for the Hisat2 aligner and the minimum quality score was set to 30 (parameter -q). Minimum read coverage and the minimum number of reads supporting the variation were defined as one read  . p-values. Differential expression analysis was performed using DESeq2  with BH-p-values was used to compare the frequencies of potential editing sites between the control and PD groups. The Fisher’s test with BH-adjusted Wilcoxon test was used to reveal the difference in the number of potential A-to-I mRNA editing sites between groups of PD and neurologically healthy people.mRNA editing is a common phenomenon in mammals, but its functional role is still largely unknown. The most prevalent type of mRNA editing is the conversion of adenosine to inosine (A-to-I editing) made by adenosine deaminases. These proteins are composed of the ADAR protein family including ADAR1 and ADAR2, which are catalytically active , and ADASeveral methods allow for the identification of RNA editing sites in transcriptome datasets . Some hahttps://www.ncbi.nlm.nih.gov/bioproject/PRJNA283498, accessed on 7 November 2020). The analyzed RNA-Seq data included on average 42,166,654 million raw paired-end reads (100 bases × 2) per sample and had been generated by the Illumina HiSeq 2000. Trimmed paired-end reads were mapped on the human reference genome (hg38) with the program hisat2 (version 2.0.5). The BAM alignments were processed with the REDItoolDenovo.py script included in the REDItools package. The script provided the list of potentially edited positions as a textual table containing coverage depth, the mean quality score, the observed base distribution, the strand, the list of observed substitutions, and the variation frequency. Only hits within gene protein-coding regions were taken into account. A set of the prefrontal cortex (BA9) samples from PD patients and neurologically healthy controls (sex and age-similar) were analyzed . Raw reahttps://gnomad.broadinstitute.org/, accessed on 9 April 2019) were excluded, and those matched to known RNA editing sites from the REDIportal were taken for further analysis. There were 160 records in the final table. The number of samples with edited bases was counted in the healthy control and PD patient samples for each position. These numbers were compared using Fisher’s test and Bonferroni multiple testing correction to identify sites showing statistically significant differential editing.The obtained dataset of potential RNA editing sites was additionally filtered: sites described earlier as potential single nucleotide variations from the gnomAD database . The differential expression analysis was performed using DESeq2 to determine relative mRNA expression of these genes (A-to-I substitutions are mediated by the adenosine deaminase family of enzymes that act on RNA (ADAR). Three ADAR genes have been identified in humans, and only two of them encode enzymes with mRNA editing activity . This statement correlates with lower levels of A-to-I editing in PD patients. Therefore, ADAR gene expression aberrations could be included in the pathogenesis of this neurodegenerative disease.Recently, RNA editing has been added to the list of common post-transcriptional modifications. It has been shown that ADAR enzymes could change A-to-I nucleotides in RNAs, affecting gene expression. Most identified editing sites reside within repeated sequences and introns . Such ed+ and/or Ca2+, regulating membrane depolarization. It is considered that decreased editing leads to exaggerated Ca2+ influx through that glutamate receptor and subsequent lower motor neuron death [A-to-I RNA editing in coding regions is quite rare, but many are associated with neuronal functions. Altered editing levels in specific coding sites have been reported for several neurological and neurodegenerative disorders such as major depression, epilepsy, schizophrenia, amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD). In most mentioned cases, A-to-I conversion is decreased due to ADAR downregulation . Generalon death . In braion death ,27. It hon death . ADAR and ADARB1 genes have been observed. These data are in accordance with previously reported evaluations of RNA editing levels in other neurodegenerative diseases. Increased expression of ADARB2 in PD samples , detected in this work, has previously been shown to be under-edited in lower grade glioma and glioblastoma [PLIN1 and GIPTC1, have recently been shown to be associated with PD. Although the obtained data need experimental verification, the first evidence for A-to-I editing in the pathogenesis of this neurodegenerative disease has been obtained.Specific substitution sites associated with the PD have also been identified. Edited sites have been found among the mRNA of genes of the subunits of the kainite and AMPA types in ionotropic glutamate receptors; it has previously been shown that ionotropic glutamate receptor antagonists could have antiparkinsonian action . Thus, ablastoma . As the blastoma . TherefoAn approach for selecting candidate A-to-I editing sites involved in PD disease was presented. This approach is based on the repurposing of open RNA-Seq data without matching genomic sequences using REDItools features. PD-specific sites with altered editing were determined. Observed lower levels of ADAR gene expression and overall decreased editing levels in PD samples could testify that mRNA modification patterns act as a part of the pathogenesis of this neurodegenerative disease.