Construction of an 11-microRNA-based signature and a prognostic nomogram to predict the overall survival of head and neck squamous cell carcinoma patients.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a fatal malignancy owing to the lack of effective tools to predict overall survival (OS). MicroRNAs (miRNAs) play an important role in HNSCC occurrence, development, invasion and metastasis, significantly affecting the OS of patients. Thus, the construction of miRNA-based risk signatures and nomograms is desirable to predict the OS of patients with HNSCC. Accordingly, in the present study, miRNA sequencing data of 71 HNSCC and 13 normal samples downloaded from The Cancer Genome Atlas (TCGA) were screened to identify differentially expressed miRNAs (DEMs) between HNSCC patients and normal controls. Based on the exclusion criteria, the clinical information and miRNA sequencing data of 67 HNSCC samples were selected and used to establish a miRNA-based signature and a prognostic nomogram. Forty-three HNSCC samples were assigned to an internal validation cohort for verifying the credibility and accuracy of the primary cohort. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore the functions of 11 miRNA target genes. RESULTS: In total, 11 DEMs were successfully identified. An 11-miRNA risk signature and a prognostic nomogram were constructed based on the expression levels of these 11 DEMs and clinical information. The signature and nomogram were further validated by calculating the C-index, area under the curve (AUC) in receiver-operating characteristic curve analysis, and calibration curves, which revealed their promising performance. The results of the internal validation cohort shown the reliable predictive accuracy both of the miRNA-based signature and the prognostic nomogram. GO and KEGG analyses revealed that a mass of signal pathways participated in HNSCC proliferation and metastasis. CONCLUSION: Overall, we constructed an 11-miRNA-based signature and a prognostic nomogram with excellent accuracy for predicting the OS of patients with HNSCC.

Urinary MicroRNAs as Potential Markers for Non-Invasive Diagnosis of Bladder Cancer.

Currently, voided urine cytology (VUC) serves as the gold standard for the detection of bladder cancer (BCa) in urine. Despite its high specificity, VUC has shortcomings in terms of sensitivity. Therefore, alternative biomarkers are being searched, which might overcome these disadvantages as a useful adjunct to VUC. The aim of this study was to evaluate the diagnostic potential of the urinary levels of selected microRNAs (miRs), which might represent such alternative biomarkers due to their BCa-specific expression. Expression levels of nine BCa-associated microRNAs (miR-21, -96, -125b, -126, -145, -183, -205, -210, -221) were assessed by quantitative PCR in urine sediments from 104 patients with primary BCa and 46 control subjects. Receiver operating characteristic (ROC) curve analyses revealed a diagnostic potential for miR-96, -125b, -126, -145, -183, and -221 with area under the curve (AUC) values between 0.605 and 0.772. The combination of the four best candidates resulted in sensitivity, specificity, positive and negative predictive values (NPV), and accuracy of 73.1%, 95.7%, 97.4%, 61.1%, and 80.0%, respectively. Combined with VUC, sensitivity and NPV could be increased by nearly 8%, each surpassing the performance of VUC alone. The present findings suggested a diagnostic potential of miR-125b, -145, -183, and -221 in combination with VUC for non-invasive detection of BCa in urine.

Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study.

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.

Pharmacological control of inflammation in wound healing.

Wound inflammation is a rapid and highly orchestrated process that significantly impacts the wound healing cascade. Consequent to injury, a series of events set off that include inflammatory, proliferation and maturation phases leading to wound closure and restoration of normal skin integrity. Stimuli causing stress to host immune system or induce inflammatory response include tissue damage and pathogenic microbial infection.Several evidences points towards the positive role of inflammation as it essential to fight against the attack of invading pathogens and to remove dead tissues from the site of injury. Besides its positive role, prolonged inflammation is injurious and may result in deregulated stages of the wound healing which may lead to excessive scarring. Achieving balance in inflammatory cascade is one of the challenging tasks for development of a wound healing drug. This review mainly focuses on the pharmacological control of inflammation by agents which critically balance the inflammatory cascade. However, none of the agent is available in the healthcare market which exclusively plays a role in wound repair. In this review we shall explore different factors or agents affecting inflammation in wound healing. This information might be helpful in designing and development new process, technologies or drugs for better management of wound care. In addition, understanding the effect of inflammation on the outcome of the healing process will serve as a significant milestone in the area of pathological tissue repair.

Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR.

The high-throughput analysis of microRNAs (miRNAs) circulating within the blood of healthy and diseased individuals is an active area of biomarker research. Whereas quantitative real-time reverse transcription polymerase chain reaction (qPCR)-based methods are widely used, it is yet unresolved how the data should be normalized. Here, we show that a combination of different algorithms results in the identification of candidate reference miRNAs that can be exploited as normalizers, in both discovery and validation phases. Using the methodology considered here, we identify normalizers that are able to reduce nonbiological variation in the data and we present several case studies, to illustrate the relevance in the context of physiological or pathological scenarios. In conclusion, the discovery of stable reference miRNAs from high-throughput studies allows appropriate normalization of focused qPCR assays.

Relevance of MicroRNAs as Potential Diagnostic and Prognostic Markers in Colorectal Cancer.

Colorectal cancer (CRC) is currently the third and the second most common cancer in men and in women, respectively. Every year, more than one million new CRC cases and more than half a million deaths are reported worldwide. The majority of new cases occur in developed countries. Current screening methods have significant limitations. Therefore, a lot of scientific effort is put into the development of new diagnostic biomarkers of CRC. Currently used prognostic markers are also limited in assessing the effectiveness of CRC therapy. MicroRNAs (miRNAs) are a promising subject of research especially since single miRNA can recognize a variety of different mRNA transcripts. MiRNAs have important roles in epigenetic regulation of basic cellular processes, such as proliferation, apoptosis, differentiation, and migration, and may serve as potential oncogenes or tumor suppressors during cancer development. Indeed, in a large variety of human tumors, including CRC, significant distortions in miRNA expression profiles have been observed. Thus, the use of miRNAs as diagnostic and prognostic biomarkers in cancer, particularly in CRC, appears to be an inevitable consequence of the advancement in oncology and gastroenterology. Here, we review the literature to discuss the potential usefulness of selected miRNAs as diagnostic and prognostic biomarkers in CRC.

Expanding the Utilization of Formalin-Fixed, Paraffin-Embedded Archives: Feasibility of miR-Seq for Disease Exploration and Biomarker Development from Biopsies with Clear Cell Renal Cell Carcinoma.

Novel predictive tools for clear cell renal cell carcinoma (ccRCC) are urgently needed. MicroRNAs (miRNAs) have been increasingly investigated for their predictive value, and formalin-fixed paraffin-embedded biopsy archives may potentially be a valuable source of miRNA sequencing material, as they remain an underused resource. Core biopsies of both cancerous and adjacent normal tissues were obtained from patients (n = 12) undergoing nephrectomy. After small RNA-seq, several analyses were performed, including classifier evaluation, obesity-related inquiries, survival analysis using publicly available datasets, comparisons to the current literature and ingenuity pathway analyses. In a comparison of tumour vs. normal, 182 miRNAs were found with significant differential expression; miR-155 was of particular interest as it classified all ccRCC samples correctly and correlated well with tumour size (R² = 0.83); miR-155 also predicted poor survival with hazard ratios of 2.58 and 1.81 in two different TCGA (The Cancer Genome Atlas) datasets in a univariate model. However, in a multivariate Cox regression analysis including age, sex, cancer stage and histological grade, miR-155 was not a statistically significant survival predictor. In conclusion, formalin-fixed paraffin-embedded biopsy tissues are a viable source of miRNA-sequencing material. Our results further support a role for miR-155 as a promising cancer classifier and potentially as a therapeutic target in ccRCC that merits further investigation.

Optimization and Standardization of Circulating MicroRNA Detection for Clinical Application: The miR-Test Case.

BACKGROUND: The identification of circulating microRNAs (miRNAs) in the blood has been recently exploited for the development of minimally invasive tests for the early detection of cancer. Nevertheless, the clinical transferability of such tests is uncertain due to still-insufficient standardization and optimization of methods to detect circulating miRNAs in the clinical setting. METHODS: We performed a series of tests to optimize the quantification of serum miRNAs that compose the miR-Test, a signature for lung cancer early detection, and systematically analyzed variables that could affect the performance of the test. We took advantage of a large-scale (>1000 samples) validation study of the miR-Test that we recently published, to evaluate, in clinical samples, the effects of analytical and preanalytical variables on the quantification of circulating miRNAs and the clinical output of the signature (risk score). RESULTS: We developed a streamlined and standardized pipeline for the processing of clinical serum samples that allows the isolation and analysis of circulating miRNAs by quantitative reverse-transcription PCR, with a throughput compatible with screening trials. The major source of analytical variation came from RNA isolation from serum, which could be corrected by use of external (spike-in) or endogenous miRNAs as a reference for normalization. We also introduced standard operating procedures and QC steps to check for unspecific fluctuations that arise from the lack of standardized criteria in the collection or handling of the samples (preanalytical factors). CONCLUSIONS: We propose our methodology as a reference for the development of clinical-grade blood tests on the basis of miRNA detection.

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.

Establishment of plasma microRNA detection method by using taqman probe based quantitative reverse transcription PCR.

MicroRNAs (miRNAs) are a kind of short non—coding RNAs that regulate gene expression at the post—transcriptional level. Recently, many studies have found that circulating miRNAs have the potential to sever as diagnostic biomarkers for many diseases. However, the methods for the quantification of circulating miRNAs still need more adjustment. In this study, we tried to establish a reliable method to quantify the plasma miRNAs. We used quantitative real—time PCR with taqman probes to detect the plasma miR—153 level. Three controls were used in this study, including two external miRNAs control from C. elegans miRNAs (cel—miR—54 and cel—miR—238) and one internal control (hsa—miR—486). All of these controls were stable in plasma and the cel—miR—238/cel—miR—54/hsa—miR—486 combination could improve the normalization process. The expression level of the target miRNA, human plasma miR—153, could be quantified accurately with taqman probes .The assay has high accuracy, high sensitivity and a large dynamic range from 100 copies to 10(13) copies in the PCR reaction. Our study provided a standardized quantification method for plasma miRNAs which might be used as biomarker in many diseases research.

mRNA and microRNA quality control for RT-qPCR analysis.

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency. RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity.

Quality assessment and data analysis for microRNA expression arrays.

MicroRNAs are small (approximately 22 nt) RNAs that regulate gene expression and play important roles in both normal and disease physiology. The use of microarrays for global characterization of microRNA expression is becoming increasingly popular and has the potential to be a widely used and valuable research tool. However, microarray profiling of microRNA expression raises a number of data analytic challenges that must be addressed in order to obtain reliable results. We introduce here a universal reference microRNA reagent set as well as a series of nonhuman spiked-in synthetic microRNA controls, and demonstrate their use for quality control and between-array normalization of microRNA expression data. We also introduce diagnostic plots designed to assess and compare various normalization methods. We anticipate that the reagents and analytic approach presented here will be useful for improving the reliability of microRNA microarray experiments.

Selection of Reference miRNAs for Relative Quantification in Buffalo (Bubalus bubalis) Blastocysts Produced by Hand-Made Cloning and In Vitro Fertilization.

Very low birth rate and a high incidence of abnormalities in offspring born from cloned embryos, which have limited the application of cloning technology on a wide scale, are believed to be because of incomplete or aberrant nuclear reprogramming. MicroRNAs (miRNAs) are involved in regulating a wide range of biological processes including reprogramming and embryonic development. Selection of suitable reference miRNAs is critical for normalization of data for accurate relative quantification of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR), which is currently the most widely used technique for quantifying miRNAs. This study was aimed at identification of reference miRNAs suitable for normalization of qRT-PCR data from blastocyst-stage buffalo embryos produced by handmade cloning and in vitro fertilization (IVF). RNA isolated from cloned and IVF blastocysts was subjected to next-generation sequencing based on which, 12 highly and most consistently expressed miRNAs, which included miR-92a, miR-423, miR-151, Let-7a, miR-103a, miR-93, miR-16b, miR-25, miR-30e, miR-101, miR-127, and miR-197, were selected as candidates for identification of suitable reference miRNAs using three statistical algorithms namely geNorm, NormFinder, and BestKeeper. Based on consensus of the three algorithms, the combination of miRNAs found to be suitable as reference miRNAs were miR-127 and miR-103 for IVF blastocysts; miR-92a and miR-103 for cloned blastocysts, and miR-103, miR-423, and miR-93 across both IVF and cloned blastocysts. The data of this study can be very useful in miRNA expression analysis of blastocyst-stage cloned and IVF embryos.

The Diagnostic and Prognostic Value of miR-200c in Gastric Cancer: A Meta-Analysis.

BACKGROUND: The role of miR-200c in gastric cancer remains controversial. This study is aimed at clarifying the diagnostic and prognostic value of miR-200c in gastric cancer through a meta-analysis. METHODS: A comprehensive literature search of PubMed, Embase, and Ovid library databases was conducted. The studies included were those conducted before December 2017. The sensitivity and specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under curve (AUC) were used to estimate the diagnostic value of miR-200c. Meanwhile, the pooled hazard ratio (HR) was used to estimate the prognostic value of miR-200c. RESULTS: For the diagnostic value of miR-200c, six studies that included 202 patients with gastric cancer and 250 normal controls were analyzed. The sensitivity, specificity, PLR, NLR, DOR, and AUC were 0.74, 0.66, 2.20, 0.40, 5.34, and 0.75, respectively. Subgroup analysis showed no significant difference in the type of the sample, method for testing miR-200c, and ethnicity among the patients. Meanwhile, for the prognostic value of miR-200c, seven studies comprising 935 patients with gastric cancer were analyzed. The pooled results showed that miR-200c expression was associated with overall survival (HR = 2.19) and disease-free survival (HR = 1.73), but not with progression-free survival (HR = 1.64) in patients with gastric cancer. There was no publication bias across the studies. CONCLUSIONS: Both serum and tissue miR-200c have moderate diagnostic accuracy in gastric cancer. miR-200c could also be used as a valuable indicator for predicting the prognosis of gastric cancer patients.

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri.

MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.

MicroRNA Signatures in Malignant Pleural Mesothelioma Effusions.

Malignant pleural mesothelioma (MPM) is an incurable cancer of the pleura that can be difficult to diagnose. Biomarkers for an easier and/or earlier diagnosis are needed. Approximately 90% of MPM patients develop a pleural effusion (PE). PEs are ideal sources of biomarkers as the fluid would almost always require drainage for diagnostic and/or therapeutic reasons. However, differentiating MPM PE from PE caused by other diseases can be challenging. MicroRNAs are popular biomarkers given their stable expression in tissue and fluid. MicroRNAs have been analysed in PE cytology samples for the diagnosis of MPM but have not been measured in frozen/fresh PE. We hypothesise that microRNAs expressed in PE are biomarkers for MPM. TaqMan OpenArray was used to analyse over 700 microRNAs in PE cells and supernatants from 26 MPMs and 21 other PE-causing diseases. In PE cells, combining miR-143, miR-210, and miR-200c could differentiate MPM with an area under the curve (AUC) of 0.92. The three-microRNA signature could also discriminate MPM from a further 40 adenocarcinomas with an AUC of 0.9887. These results suggest that the expression of miR-143, miR-210, and miR-200c in PE cells might provide a signature for diagnosing MPM.

Clinical significance and prognostic role of hypoxia-induced microRNA 382 in gastric adenocarcinoma.

Hypoxia and angiogenesis are critical components in the progression of solid cancer, including gastric cancers (GCs). miR-382 has been identified as a hypoxia-induced miR (hypoxamiR), but the clinical significance in GCs has not been identified yet. To explore the clinical and prognostic importance of miR-382 in GCs, the surgical specimens of 398 patients with GCs in KNU hospital in Korea, the total of 183 patients was randomly selected using simple sampling methods and big data with 446 GCs and 45 normal tissues from the data portal (https://portal.gdc.cancer.gov/) were analysed. Expression of miR-382 as well as miR-210, as a positive control hypoxamiR by qRT-PCR in histologically malignant region of GCs showed significantly positive correlation (R = 0.516, p<0.001). High miR-210 and miR-382 expression was significantly correlated with unfavorable prognosis including advanced GCs (AGC), higher T category, N category, pathologic TNM stage, lymphovascular invasion, venous invasion, and perinueral invasion, respectively (all p<0.05). In univariate analysis, high miR-210 expression was significantly associated with worse overall survival (OS) (p = 0.036) but not high miR-382. In paired 60 gastric normal and cancer tissues, miR-382 expression in cancer tissues was significantly higher than normal counterpart (p = 0.003), but not miR-210 expression. However, by increasing the patient number from the big data analysis, miR-210 as well as miR-382 expression in tumor tissues was significantly higher than the normal tissues. Our results suggest that miR-382, as novel hypoxamiR, can be a prognostic marker for advanced GCs and might be correlated with metastatic potential. miR-382 might play important roles in the aggressiveness, progression and prognosis of GCs. In addition, miR-382 give a predictive marker for progression of GCs compared to the normal or preneoplastic lesion.

Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology.

BACKGROUND: During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising tool for the development of new diagnostic and prognostic markers. However, most human tumour samples for which long term clinical records are available exist only as formalin-fixed paraffin-embedded specimens. Therefore, the aim of this study was to examine the feasibility of microRNA profiling studies in routinely processed formalin-fixed paraffin-embedded human breast cancer specimens using fluorescence labelled bead technology. RESULTS: A statistically highly significant correlation (Spearman r: 0.78 - 0.90, p < 0.0001) was observed for the expression of 319 microRNAs in routinely processed FFPE breast cancer specimens and paired fresh frozen tissue samples (n = 5). Results were confirmed in a larger series analyzing a selection of 10 microRNAs reported to be deregulated in breast cancer (n = 12). The expression pattern of 3 microRNAs was independently validated in this cohort using real-time RT-PCR technology. CONCLUSION: Comprehensive microRNA expression patterns can be reliably derived from routinely processed FFPE breast cancer specimens using fluorescence labelled bead technology.

Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Small Nucleolar RNA Score: An Assay to Detect Formalin-Overfixed Tissue.

Although there are millions of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown fixation conditions. We analyzed FFPE tissue biospecimens as part of the NCI Biospecimen Preanalytical Variables (BPV) program to identify microRNA (miRNA) markers for fixation time. miRNA was extracted from kidney and ovary tumor FFPE blocks (19 patients, cold ischemia ≤2 hours) with 6, 12, 24, and 72 hours fixation times, then analyzed using the WaferGen SmartChip platform (miRNA chip with 1036 miRNA targets). For fixation time, principal component analysis of miRNA chip expression data separated 72 hours fixed samples from 6 to 24 hours fixed samples. A set of small nuclear RNA (snRNA) targets was identified that best determines fixation time and was validated using a second independent cohort of seven different tissue types. A customized assay was then developed, based on a set of 24 miRNA and snRNA targets, and a simple "snoRNA score" defined. This score detects FFPE tissue samples with fixation for 72 hours or more, with 79% sensitivity and 80% specificity. It can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be of limited robustness to formalin overfixation.