RNA Integrity Number Values Research Articles

Effects of Warm Ischemia Time, Cryopreservation, and Grinding Methods on RNA Quality of Mouse Kidney Tissues.

Background: High-quality RNA extraction from tissue samples is of key importance for scientific research and translational medicine. Tissue collection and preparation may affect RNA quality. In this study, we investigated effects of warm ischemia time, cryopreservation, and grinding methods on RNA quality. Methods: Total RNA was extracted from mouse kidney tissues with warm ischemia times of 0, 30, 60, 90, and 120 minutes. Half of the tissues were used to extract RNA immediately, while the others were cryopreserved in the vapor phase of liquid nitrogen for 6 months before RNA extraction. A mortar, homogenizer, and tissue lyser were used to grind tissues. RNA was extracted by TRIzol, and RNA integrity was assessed by the RNA integrity number (RIN) value. Results: For fresh tissues and frozen tissues with warm ischemia time within 60 minutes, RIN values were above 7.0 and remained above 6.0 with warm ischemia time within 120 minutes. For the same warm ischemia time, RIN values of frozen tissues were slightly lower than those of fresh tissues. No significant RIN value alterations were observed among grinding methods, but for RNA extraction efficiency, a mortar was much less efficient than the homogenizer or tissue lyser. For frozen tissues, RNA tended to degrade within 8 minutes at room temperature. Conclusions: Mouse kidney tissues with a warm ischemia time within 120 minutes are suitable for general RNA-related research. For tissues with a warm ischemia time within 60 minutes, cryopreservation may not affect RNA quality. The duration of frozen tissues held at room temperature before grinding affects the integrity of RNA, while grinding methods do not affect RNA integrity.

A simple phenol-free isolation method for high-quality RNA from bilberry

High-quality RNA is required for accurate gene expression and transcriptome analysis. The current methods of RNA extraction from berry fruits are either time-consuming or expensive. To simplify the conventional phenol-chloroform based RNA extraction method, we modified the protocol with less steps as well as the removal of the use of phenol. In this protocol, the extraction buffer is composed of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and Dithiothreitol (DTT). The method facilitates efficient removal of polysaccharides and phenolic compounds from both fruit pulp and fruit peel. Additionally, the protocol is phenol-free and less toxic than traditional phenol-containing method. High-quality RNA, with RNA Integrity Number value > 8, isolated by this method is applicable for RNA sequencing and qPCR. Only 3–4 working hours are required for one batch of RNA isolation.•Our method replaces the use of phenol-chloroform with chloroform, making the extraction less toxic.•The bilberry friut RNA is of high-quality and purity with less time input.

Effects of brain tissue section processing and storage time on gene expression

The pre-processing of samples is important factors that affect the results of the RNA-sequencing (RNA-seq) data. However, the effects of frozen sections storage conditions on the integrity of RNA and sequencing results haven’t been reported. The study of frozen section protection schemes can provide reliable experimental results for single-cell and spatial transcriptome sequencing. In this study, RNA was isolated to be studied for RNA from brain section at different temperatures (RT: room temperature, −20 °C) and storage time (0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h, 7day, 3week, 6month). The stability of reference genes was validated using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the storage at room temperature significantly affected RNA integrity number (RIN), and the RIN value was lower with the prolongation of storage, while the storage at −20 °C exerted less effect on the RIN value. Cresyl violet staining for brain tissue sections had little effect on RNA integrity. 1925, 899 and 3390 differential expression genes (DEGs) were screened at 2 h, 4 h and 8 h at room temperature, respectively. A total of 892, 478 and 619 genes were shown to be differentially expressed at −20 °C for 7d, 3w and 6 m, respectively. Among them, the expression of glycoprotein m6a (Gpm6a), calmodulin 1 (Calm1), calmodulin 1 (Calm2), thymosin, beta 4, X chromosome (Tmsb4x), ribosomal protein S21 (Rps21) and so on were correlated with RNA quality. According to the expression stability of 4 reference genes (Actb: beta-actin; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; 18S: 18S ribosomal; Hprt1: hypoxanthine phosphoribosyltransferase 1), 18S is the most stable reference gene in the brain. In conclusion, the storage temperature and time of frozen sections have significant effects on RNA integrity and sequencing results. But there are still some genes that are stable and not affected by worsening of overall RNA integrity ie the decrease of RIN value. In addition, 1% cresyl violet staining can protect RNA.

Low RIN Value for RNA-Seq Library Construction from Long-Term Stored Seeds: A Case Study of Barley Seeds.

Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low RNA integrity number (RIN). Here, barley seeds that have almost completely lost their viability as a result of long-term storage were used. As a control, fully viable seeds obtained in the course of field regeneration were used. The effectiveness of protocols dedicated to RNA samples with high and low RIN values was compared. The experiment concluded that library construction from low viable or long-term stored seeds with degraded RNA (RIN < 3) should be carried out with extraordinary attention due to the possibility of uneven degradation of different RNA fractions.

Stress–response relationships related to ageing and death of orthodox seeds: a study comparing viability and RNA integrity in soya bean (Glycine max) cv. Williams 82

AbstractCharacterizing non-lethal damage within dry seeds may allow us to detect early signs of ageing and accurately predict longevity. We compared RNA degradation and viability loss in seeds exposed to stressful conditions to quantify relationships between degradation rates and stress intensity or duration. We subjected recently harvested (‘fresh’) ‘Williams 82’ soya bean seeds to moisture, temperature and oxidative stresses, and measured time to 50% viability (P50) and rate of RNA degradation, the former using standard germination assays and the latter using RNA Integrity Number (RIN). RIN values from fresh seeds were also compared with those from accessions of the same cultivar harvested in the 1980s and 1990s and stored in the refrigerator (5°C), freezer (−18°C) or in vapour above liquid nitrogen (−176°C). Rates of viability loss (P50−1) and RNA degradation (RIN⋅d−1) were highly correlated in soya bean seeds that were exposed to a broad range of temperatures [holding relative humidity (RH) constant at about 30%]. However, the correlation weakened when fresh seeds were maintained at high RH (holding temperature constant at 35°C) or exposed to oxidizing agents. Both P50−1 and RIN⋅d−1 parameters exhibited breaks in Arrhenius behaviour near 50°C, suggesting that constrained molecular mobility regulates degradation kinetics of dry systems. We conclude that the kinetics of ageing reactions at RH near 30% can be simulated by temperatures up to 50°C and that RNA degradation can indicate ageing prior to and independent of seed death.

Optimising the sampling procedure for forensic investigation of bruises on pigs

BackgroundHuman-inflicted bruises on pigs are a violation of the law and affected tissue is regularly sent for forensic investigation. The authors aimed to evaluate the variation in inflammation within and...

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples.

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality.

Background: Circular RNAs (circRNAs) are a new class of RNAs with medical significance. Compared to that of linear mRNA transcripts, the stability of circRNAs against degradation owing to their circular structure is considered advantageous for their use as biomarkers. As systematic studies on the stability of circRNAs depending on the RNA integrity, determined as RNA integrity number (RIN), in clinical tissue samples are lacking, we have investigated this aspect in the present study under model and clinical conditions.Methods: Total RNA isolated from kidney cancer tissue and cell lines (A-498 and HEK-293) with different RIN after thermal degradation was used in model experiments. Further, RNA isolated from kidney cancer and prostate cancer tissue collected under routine surgical conditions, representing clinical samples with RIN ranging from 2 to 9, were examined. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis of several circRNAs (circEGLN3, circRHOBTB3, circCSNK1G3, circRNA4, and circRNA9), their corresponding linear counterparts, tissue-specific reference genes, and three microRNAs (as controls) was performed. The quantification cycles were converted into relative quantities and normalized to the expression of specific reference genes for the corresponding tissue. The effect of RIN on the expression of different RNA entities was determined using linear regression analysis, and clinical samples were classified into two groups based on RIN greater or lesser than 6.Results: The results of model experiments and clinical sample analyses showed that all relative circRNA expression gradually decreased with reduction in RIN values. The adverse effect of RIN was partially compensated after normalizing the data and limiting the samples to only those with RIN values > 6.Conclusions: Our results suggested that circRNAs are not stable in clinical tissue samples, but are subjected to degradative processes similar to mRNAs. This has not been investigated extensively in circRNA expression studies, and hence must be considered in future for obtaining reliable circRNA expression data. This can be achieved by applying the principles commonly used in mRNA expression studies.

Extraction of high-quality RNA from mouse pancreatic tumors

Extraction of high-quality RNA from pancreatic tumors for sequencing purposes is technically challenging, as the pancreas is an organ rich in ribonucleases. The majority of the established RNA isolation protocols for use with primary pancreatic tissue involve perfusion of RNA stabilizing reagent into the pancreatic tissue to protect RNA integrity before extraction. However, the additional time needed for this procedure can actually lead to further RNA degradation. We optimized a protocol suitable for high quality RNA isolation from mouse pancreatic tumors that is a simple, fast, and inexpensive modification of existing methods, combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction. Through this procedure, the mean RNA Integrity Number value obtained for RNA isolated from pancreatic tumors was 9.0, and was reproducibly suitable for RNAseq and qPCR.•a protocol suitable for high quality RNA isolation from mouse pancreatic tumors as well as normal pancreas•combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction

Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods.

Gene expression profiling using blood samples is a valuable tool for biomarker discovery in clinical studies. Different whole blood RNA collection and processing methods are highly variable and might confound comparisons of results across studies. The main aim of the current study is to compare how blood storage, extraction methodologies, and the blood components themselves may influence gene expression profiling. Whole blood and peripheral blood mononuclear cell (PBMC) samples were collected in triplicate from five healthy donors. Whole blood was collected in RNAgard® and PAXgene® Blood RNA Tubes, as well as in collection tubes with anticoagulants such as dipotassium ethylenediaminetetraacetic acid (K2EDTA) and Acid Citrate Dextrose Solution A (ACD-A). PBMCs were separated using sodium citrate Cell Preparation Tubes (CPT™), FICOLL™, magnetic separation, and the LeukoLOCK™ methods. After blood collection, the LeukoLOCK™, K2EDTA and ACD-A blood tubes were shipped overnight using cold conditions and samples from the rest of the collection were immediately frozen with or without pre-processing. The RNA was isolated from whole blood and PBMCs using a total of 10 different experimental conditions employing several widely utilized RNA isolation methods. The RNA quality was assessed by RNA Integrity Number (RIN), which showed that all PBMC procedures had the highest RIN values when blood was stabilized in TRIzol® Reagent before RNA extraction. Initial data analysis showed that human blood stored and shipped at 4°C overnight performed equally well when checked for quality using RNA integrity number when compared to frozen stabilized blood. Comparisons within and across donor/method replicates showed signal-to-noise patterns which were not captured by RIN value alone. Pathway analysis using the top 1000 false discovery rate (FDR) corrected differentially expressed genes (DEGs) showed frozen vs. cold shipping conditions greatly impacted gene expression patterns in whole blood. However, the top 1000 FDR corrected DEGs from PBMCs preserved after frozen vs. cold shipping conditions (LeukoLOCK™ preserved in RNAlater®) revealed no significantly affected pathways. Our results provide novel insight into how RNA isolation, various storage, handling, and processing methodologies can influence RNA quality and apparent gene expression using blood samples. Careful consideration is necessary to avoid bias resulting from downstream processing. Better characterization of the effects of collection method idiosyncrasies will facilitate further research in understanding the effect of gene expression variability in human sample types.

A New Technique in Fresh Prostate Cancer Tissue Biobanking Based on MRI-Transrectal Ultrasound Fusion Biopsy

To present and validate a new technique for biobanking fresh-frozen prostate cancer tissue based on MRI-transrectal ultrasound fusion biopsy. From August 2014 to August 2016, patients with elevated levels of PSA and at least 1 suspicious lesion on MRI were invited to this study. Each MRI-suspicious lesion was biopsied repeatedly for at least 2 cores in the same location. These repeated cores were labelled A/A', B/B', etc. The A/B cores were submitted for histologic assessment, and the corresponding A'/B' cores were stored in an -80°C freezer for biobanking. Sixty biobanked samples were processed for histologic assessment to compare their pathologic parameters with their corresponding paraffin samples. Another 20 biobanked samples were processed for RNA quality evaluation. Fifty-six of the 60 selected banking samples matched their corresponding paraffin samples for benign vs malignant diagnosis, leading an overall concordance rate of 93.3%. There was no significant difference between banking samples and the corresponding paraffin samples in cancer percentage and Gleason score. The RNA Integrity Number value ranged from 6.8 to 9.3 (mean 7.89). The current study demonstrates that the histologic identity of the banked prostate biopsy sample can be accurately predicted by its corresponding paraffin samples. MRI-TRUS fusion biopsy based biobanking method is highly efficient, timesaving, and has high quality tissues both at the histologic and RNA integrity levels.

Quality Control of RNA Extracted from PAXgene Blood RNA Tubes After Different Storage Periods.

Background: The PAXgene Blood RNA tube is used to protect RNA of whole blood, and the stability of RNA in this tube has already been reported. However, there are few reports on the quality of RNA from long-term preservation of these tubes. Materials and Methods: Our biobank conducted quality control of RNA extracted from the tubes after varying storage periods. A total of 300 blood samples of renal disease patients were randomly selected at each time point (from 1 month to 7 years). Results: Median RNA yields were 3.46 (2.65-4.87) μg, 4.34 (3.20-5.87) μg, 4.77 (2.88-6.29) μg, 4.19 (2.65-6.26) μg, 3.85 (2.43-6.13) μg, and 3.21 (1.85-6.61) μg at 1 month, 1, 2, 3, 6, and 7 years, respectively. There were no significant differences in RNA yields among all the storage periods. A260/280 ratios were 2.02 ± 0.04, 2.05 ± 0.04, 2.05 ± 0.04, 2.08 ± 0.03, 2.12 ± 0.10, and 2.11 ± 0.05, all of which were ≥1.8. However, A260/280 of the samples stored for 6 and 7 years had a rising trend, compared with the other time points (p < 0.05). Median RNA integrity number (RIN) values were 8.4 (7.6-9.1), 8.3 (7.7-8.9), 8.3 (7.8-8.8), 8.5 (8.3-8.9), 7.6 (7.1-8.1), and 7.9 (7.2-8.3) at each time point. Lower RIN values were found at 6 and 7 years compared with the other storage periods (p < 0.05). The rates of RIN values ≥7.0 were 92%, 84%, 96%, 92%, 86%, and 84%, which exhibited no differences across all the storage periods. In addition, the yields and RIN values of RNA from samples with blood clots were significantly lower than those without (p < 0.001). Conclusions: The quantity and quality of RNA extracted from PAXgene Blood RNA tubes are stable throughout cryopreservation for 7 years.

Acute Effects of Whole Egg Consumption on Circulating MicroRNAs in Sprague Dawley Animals (P15-001-19)

ObjectivesMicroRNAs, small ∼22 nt non-coding RNAs, have a functional role in post-transcriptional regulation of gene expression. We have shown that whole egg-based diets reduce weight gain despite increased food intake compared to casein-based diets in Zucker-Diabetic Fatty (ZDF) rats. We hypothesized that the microRNA content of whole egg may underlie the differential impact on weight gain in ZDF rats.Our first objective was to characterize the circulating microRNA content in response to feeding whole egg-based diets over an 8 hour period. Our second objective was to identify and characterize the microRNAs present in whole egg- and casein-based diets. MethodsMale Sprague Dawley rats (n = 12; 6 weeks of age) were meal-trained to consume 4 grams of feed in one hour. After one week of meal-training, animals were fasted overnight for 10 hours (h) with water ad libitum followed by controlled feeding of either an AIN93G casein-based diet or isocaloric AIN93G diet containing whole egg (20% egg protein, w/w). At 8 weeks of age, serum was collected via the tail vein at 0, 2, 4, 6, and 8 h following refeeding. Serum microRNA was extracted using a RNA SPLIT kit and prepared for sequencing on an Illumina HiSeq 3000 using a smallRNA Library Prep. Five RNA extraction methods were tested on 6 samples of each diet: 1) Trizol (Invitrogen), 2) Trizol + Directzol (Zymogen), 3) Trizol followed by a second extraction using a fatty acid RNA extraction kit (Norgen), 4) mirPremier (Sigma-Aldrich), 5) ISOLATE II RNA (Bioline). RNA 260/280 ratios were assessed via Nanodrop and RNA integrity was assessed on an Agilent Bioanalyzer. ResultsThe greatest total RNA recovery (ng/μL) and most accurate 260/280 absorbance ratios were achieved using Trizol. The following concentrations of extracted RNA and their respective ratios (means ± SD) were achieved for each dietary component: casein diet (460.9 ± 528.1; 1.56 ± 0.12) and whole egg diet (394.3 ± 292.8; 1.55 ± 0.16), whole egg (73.4 ± 20.3; 1.15 ± 0.23), corn oil (67.5 ± 20.0; 1.41 ± 0.04), and cornstarch (247.12 ± 110.34; 1.64 ± 0.13). Total RNA recovered from the diets had an RNA integrity number (RIN) of 0 and microRNA concentrations were undetected. ConclusionsDespite successful total RNA recovery from the diets, all samples had a RIN value of 0, which is not optimal for downstream sequencing. Currently, microRNAs from plasma are being sequenced to determine if varying diets had differential effects on circulating microRNA expression. Funding SourcesPresidential Interdisciplinary Research Seed Grant Program, Iowa State University.

An efficient method for extracting next-generation sequencing quality RNA from liver tissue of recalcitrant animal species.

The next-generation RNA sequencing technologies expedite the discovery of a large number of novel transcripts and genes associated with various pathophysiological conditions. These technologies involve poly(A) enrichment, which in turn requires micrograms of high-quality total RNA. Unfortunately, the available RNA isolation approaches produce poor quality total RNA from difficult-to-isolate animal tissues, such as the liver with high glycogen content. Moreover, the extraction efficiencies of these approaches vary significantly depending on the animal species. To address this challenge, we optimized a three-step protocol for the extraction of high-yield and high-quality total RNA from the liver tissue (LT). The procedure effectively resolved the problem of glycogen coprecipitation by its stepwise removal. No signs of RNA degradation on gel electrophoresis analysis and RNA integrity number values ≥8.5 indicated that the extracted RNA is suitable for downstream processing, such as poly(A) enrichment and transcriptome profiling. To demonstrate the robustness of the novel protocol, a comparison was made with other currently available RNA extraction approaches from diverse resources. Whereas other protocols yielded partially degraded bands with either decreased or reversed ribosomal RNA (rRNA) ratio, our protocol yielded intact rRNA with a ratio of 2.0 ± 0.1. This optimized protocol was also successfully followed for other animal tissues, such as the bone and muscles. In conclusion, the study has described a highly efficient method for the next-generation sequencing quality RNA isolation from LT across a broad range of animal species, with extended applicability to other difficult-to-isolate tissues.

Temporal RNA Integrity Analysis of Archived Spaceflight Biological Samples

Abstract In spaceflight experiments, model organisms are used to assess the effects of microgravity on specific biological systems. In many cases, only one biological system is of interest to the Principal Investigator. To maximize the scientific return of experiments, the remaining spaceflight tissue is categorized, documented, and stored in the biobank at NASA Ames Research Center, which is maintained by the Ames Life Science Data Archive (ALSDA). The purpose of this study is to evaluate the state of a sample set of tissues from the ALSDA biobank. Garnering information – such as downstream functional analysis for the generation of omics datasets – from tissues is, in part, dependent on the state of sample preservation. RNA integrity number (RIN) values have been calculated for rodent liver tissues that were part of scientific payloads returned from the International Space Station (ISS). Rat livers from Spacelab Life Sciences 1 (SLS-1) and mouse livers from Commercial Biomedical Test Module 3 (CBTM-3), Rodent Research 1 (RR1), and Rodent Research 3 (RR3) were tested. It was found that mean RIN values from CBTM-3, RR1, and RR3 were suitable for downstream functional analysis (RIN &gt; 5) while the mean RIN value for SLS-1 was not (RIN = 2.5 ± 0.1). Information from this study lays the foundation for future efforts in determining the types of assays that are most appropriate for different tissues in the ALSDA biobank and similar preservation facilities, which would aid in shaping the design of experiments.

Biobanking of Fresh-Frozen Gastric Cancer Tissues: Impact of Long-Term Storage and Clinicopathological Variables on RNA Quality.

Molecular research is increasingly dependent on high-quality biobanked biospecimens. Preanalytical variables in tissue processing and preservation may influence the RNA quality and research results. Hence, the effect of long-term storage and clinicopathological parameters on RNA quality needs to be elucidated. Ninety gastric cancer tissue samples were collected and fresh-frozen in a -80°C freezer for 12 years (2006-2017). The histology was assessed and RNA integrity number (RIN) was detected by an Agilent 2100 Bioanalyzer. The impact of storage duration on RNA integrity and histomorphology was analyzed. The difference between RIN values and clinical variables was analyzed. Correlations between pathological parameters such as tumor cell percentage, stroma percentage, necrosis extent, cellularity, and RIN were assessed, respectively. Long-term storage at -80°C for 12 years did not adversely affect RNA integrity and histomorphology. RNA integrity was also not influenced by tumor location, estimated blood loss, cold/warm ischemia time, and surgical approach. However, RIN values were significantly correlated with the tumor cell percentage and stroma percentage. Gastric cancer tissues with higher tumor cell percentage or lower stroma percentage had higher RIN values. RNA quality of fresh-frozen gastric cancer tissues is influenced by clinical and histological parameters. Standard tissue collection procedure and histological quality control remain essential for tissue biobanking.

Abstract 862: Effects of fixation and antigen retrieval on the RNA integrity number

Abstract The effect of fixation and antigen retrieval (AR) on RNA as measured by the RNA integrity number (RIN) has not been adequately explored. RIN is an index (pseudo-measure) used to measure stability of mRNA; however, RIN is an actual measure of the stability of ribosomal RNA and not an actual measure of mRNA. RIN values range from 1 for highly degraded to 10 for intact RNA. Most surgical specimens are fixed in 10% neutral buffered formalin (3.7% formaldehyde) because of its ability to preserve morphological detail and prevent degradation. However, formaldehyde fixation causes changes in proteins through formation of crosslinks, which masks the immunorecognition of some antigens. In immunohistochemical evaluations, antigen retrieval (AR) approaches e.g., boiling of tissue sections under pressure are used to reverse these effects of fixation. It is believed that fresh or snap frozen specimens serve as most the suitable source of intact mRNA, DNA and other macromolecules for molecular analysis. However, more recent studies using rt-Q PCR indicate that RNA and DNA can be measured in formalin fixed paraffin embedded (FFPE) tissue samples which usually have RIN values of 3 or less. The question arises as to what is the usefulness of RIN in FFPE tissues. This study used three cancer cell lines DU145 (prostate), MDA231 (breast) and S2VP10 (pancreas) to examine the relationship between 10% NBF fixation, RIN and AR. The first objective was to analyze whether the duration of fixation of cells in 10% NBF correlated with decreases in RIN. Three fixation times i.e., 5 minutes, 20 minutes and 120 minutes were studied. The second objective was to investigate the effects of two AR approaches on RIN. Two AR conditions at pH 6 and pH 9 were used on the same cell lines and fixation times. For each cell line and treatment i.e., no fixation, or fixation at different times (with and without AR), RNA was extracted and RIN analyzed using the Agilent 2100 Bioanalyzer. RIN values obtained from the Agilent 2100 Bioanalyzer were statistically analyzed. T-tests show that the large decreases to about 3 in RIN values after fixation (5, 20 and 120 minutes) are statistically significant for all cell lines. All p-values are less than 0.0001. This study confirms that fixation of cells in 10% NBF lowers RIN to about 3 the value observed for fixed tissues. RIN did not vary significantly with time of fixation in 10% NBF and RIN did not change from the fixed values after either condition of AR. Thus RIN is not a useful measure of RNA stability in 10% NBF fixed tissue. Citation Format: Dennis Otali, Maitlyn Mullen, He Qinghua, Denise Oelschlager, William E. Grizzle. Effects of fixation and antigen retrieval on the RNA integrity number [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 862.

Impact of RNA integrity and blood sample storage conditions on the gene expression analysis.

BackgroundThe reliability of RNA sequencing (RNA-seq) output is affected by the quality of RNAs, which is in turn dependent on the quality of samples. Therefore, the purposes of this study were to reconsider the threshold of the RNA integrity number (RIN) and propose a simple and efficient storage scheme of blood samples for RNA-seq.Patients and methodsThe RNAs were extracted from blood samples that were stored at different conditions and used for sequencing. The bioinformatic analyses were performed to evaluate the impact of RNA integrity and blood sample storage conditions on the gene expression analysis.ResultsOur outcomes showed that the samples with RIN values more than 5.3 scarcely affected the quantitative results of RNA-seq, and the influence of inherent cellular physiological processes on RNA-seq output could be negligible.ConclusionThe blood samples stored at 4°C within 7 days with RIN values more than 5.3 were available for RNA-seq.

Method Optimization for Extracting High-Quality RNA From the Human Pancreas Tissue

Nucleic acid sequencing is frequently used to determine the molecular basis of diseases. Therefore, proper storage of biological specimens is essential to inhibit nucleic acid degradation. RNA isolated from the human pancreas is generally of poor quality because of its high concentration of endogenous RNase. In this study, we optimized the method for extracting high quality RNA from paired tumor and normal pancreatic tissues obtained from eight pancreatic cancer patients post-surgery. RNA integrity number (RIN) was checked to evaluate the integrity of RNA, we tried to extract the RNA with an RIN value of 8 or higher that allows for the latest genetic analysis. The effect of several parameters, including the method used for tissue lysis, RNAlater treatment, tissue weight at storage, and the time to storage after surgical resection, on the quantity and quality of RNA extracted was examined. Data showed that the highest quantity of RNA was isolated using a combination of manual and mechanical methods of tissue lysis. Additionally, sectioning the tissues into small pieces (<100 mg) and treating them with RNAlater solution prior to storage increased RNA stability. Following these guidelines, high quality RNA was obtained from 100% (8/8) of tumor tissues and 75% (6/8) of normal tissues. High-quality RNA was still stable under repeated freezing and thawing. The application of these results during sample handling and storage in clinical settings will facilitate the genetic diagnosis of diseases and their subsequent treatment.

Effect of Postmortem Interval and Years in Storage on RNA Quality of Tissue at a Repository of the NIH NeuroBioBank

Brain tissue from 1068 donors was analyzed for RNA quality as a function of postmortem interval (PMI) and years in storage. Approximately 83% of the cortical and cerebellar samples had an RNA integrity number (RIN) of 6 or greater, indicating their likely suitability for real-time quantitative polymerase chain reaction research. The average RIN value was independent of the PMI, up to at least 36 hours. The RNA quality for specific donated brains could not be predicted based on the PMI. Individual samples with a low PMI could have a poor RIN value, while a sample with a PMI over 36 hours may have a high RIN value. The RIN values for control brain donors, all of whom died suddenly and unexpectedly, were marginally higher than for individuals with clinical brain disorders. Polymerase chain reaction (PCR) analysis of samples confirmed that RIN values were more critical than PMI for determining suitability of tissue for molecular biological studies and samples should be matched by their RIN values rather than PMI. Importantly, PCR analysis established that tissue stored up to 23 years at −80°C yielded high-quality RNA. These results confirm that postmortem human brain tissue collected by brain and tissue banks over decades can serve as high quality material for the study of human disorders.