Experience of quantity and quality of DNA and RNA extraction from limited pediatric blood samples: A comparative analysis of automated and manual kit-based method.

INTRODUCTION: Optimal DNA and RNA quantity and purity is essential for downstream molecular biology experimentation and to avoid re-processing of sample. Despite availability of different kits and automated systems for nucleic acid isolation there is limited data on their performance evaluation, more so with pediatric blood samples, that are usually compromised in quantity. Hence, we evaluated the performance of automated QIAcube platform using pediatric blood samples in parallel with manual Qiagen extraction kits. MATERIALS AND METHODS: : A total of 500 samples were analyzed based on groups of PBMC and direct blood input. The isolated DNA and RNA were surveyed for quantity and quality tests by spectrophotometric and downstream analysis. RESULTS: : There was no significant difference in the DNA quantity (ng/ul) between manual and automated method based on similar sample input but quality (260/280) was significantly better with the QIAcube platform when direct blood and or PBMCs were used for extraction respectively (1.82 ± 004 Vs. 1.84.002; P-0.000008 and 1.859 ± 005 Vs. 1.843 ± 0.003; P-0.02). Moreover, the standard error mean was low for both quantity and quality in the QIAcube method suggesting uniformity. Comparison of quality assessment by spectrophotometer and qubit fluorimeter showed that QIAcube sheared DNA less (P- 0.038) as compared to manual method (P-0.013). Also, time taken to process the samples in QIAcube was 23% less than the kit-based method. CONCLUSION: Overall analysis of QIAcube platform suggests that it yields more better, uniform, and less-sheared quality of nucleic acid in a relatively less time as compared to manual extraction kits.

PEPPRO: quality control and processing of nascent RNA profiling data.

Nascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using a cluster, providing a portable first step for genomic nascent RNA analysis.

Overcoming the Challenges of High Quality RNA Extraction from Core Needle Biopsy.

The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA's compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology.

Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue.

Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

Directrices de Laboratorio para la Detección y el Diagnóstico de la Infección con el Virus COVID-19 / Laboratory Guidelines for the Detection and Diagnosis of COVID-19 Virus Infection

Los coronavirus son un grupo de virus ARN altamente diversos de la familia Coronaviridae que se dividen en 4 géneros: alfa, beta, gamma y delta, y que causan enfermedades de leves a graves en humanos y animales (1-3). Existen coronavirus humanos endémicos como los alfacoronavirus 229E y NL63 y los betacoronavirus OC43 y HKU1 que pueden causar enfermedades de tipo influenza o neumonía en humanos (1, 3). Sin embargo, dos coronavirus zoonóticos que causan enfermedades graves en humanos han emergido: el coronavirus del Síndrome respiratorio agudo grave (SARS-CoV) en 2002-2003 y el coronavirus del Síndrome respiratorio de Oriente Medio (MERS-CoV) (1-5). En enero de 2020, el agente etiológico responsable de un grupo de casos de neumonía grave en Wuhan, China, fue identificado como un nuevo betacoronavirus, distinto del SARS-CoV y MERS-CoV (6). El 11 de febrero de 2020, el Comité Internacional de Taxonomía de Virus (ICTV) anunció la denominación del virus como coronavirus del síndrome respiratorio agudo grave 2 (SARS-CoV-2) (7), mientras que, el mismo día, la OMS nombró la enfermedad como enfermedad por coronavirus COVID-19 (8). Para fines de comunicación, haremos referencia a este virus como "el virus responsable de COVID-19" o "el virus COVID-19". La secuencia genómica completa de este nuevo agente está disponible y se han desarrollado diferentes protocolos de detección (9). A la luz de la circulación actual de COVID-19 en la región de las Américas, la Organización Panamericana de la Salud / Organización Mundial de la Salud (OPS / OMS) recomienda a los Estados Miembros garantizar la identificación oportuna de casos sospechosos, la toma y el envío de muestras a los laboratorios de referencia, y la implementación de protocolos de detección molecular, según la capacidad del laboratorio.

The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. METHODS: Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. RESULTS: Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63-0.66) and between technical replicates (median expression difference 0.13-0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31-0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91-0.96). CONCLUSIONS: The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Optimized Biobanking Procedures for Preservation of RNA in Tissue: Comparison of Snap-Freezing and RNAlater-Fixation Methods.

Introduction: Personalized treatment, supported by biomarkers, would improve survival of ovarian cancer patients. RNA molecules are potentially important biomarkers. The Danish CancerBiobank provides an infrastructure for handling and storage of biological material, including RNA, from Danish cancer patients. The aim of this study was to investigate the effects of handling-time and fresh-freezing versus RNAlater® fixation on RNA degradation in solid tissue from pelvic mass samples. Materials and Methods: We evaluated RNA quality in surgical tissue from patients with a pelvic mass. Corresponding samples were either fresh-frozen or fixed in RNAlater, at eight different time points after the surgery. Integrity was measured using a bioanalyzer, and the amount and quality were further investigated by quantitative reverse transcription-polymerase chain reaction measuring the expression of housekeeping genes B2M and HPRT1. Results: Our results show that tissue RNA is stable up to at least 180 minutes after the surgery, as the quality was comparable to the quality of RNA handled immediately. Likewise, patient RNA was of acceptable quality after both fresh-frezing and RNAlater fixation, but RNAlater fixation was slightly more effective for RNA preservation. Discussion and Conclusion: Our data suggest that RNA in pelvic mass samples is relatively stable. Knowledge about RNA stability is an important prerequisite for research in RNA biomarkers, where the challenge is to balance the need for careful RNA handling and storage with the need for effective large-scale biobanking in a busy clinical setting where patient treatment is the main priority.

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.

Expression Concordance of 325 Novel RNA Biomarkers between Data Generated by NanoString nCounter and Affymetrix GeneChip.

BACKGROUND: With the development of new drug combinations and targeted treatments for multiple types of cancer, the ability to stratify categories of patient populations and to develop companion diagnostics has become increasingly important. A panel of 325 RNA biomarkers was selected based on cancer-related biological processes of healthy cells and gene expression changes over time during nonmalignant epithelial cell organization. This "cancer in reverse" approach resulted in a panel of biomarkers relevant for at least 7 cancer types, providing gene expression profiles representing key cellular signaling pathways beyond mutations in "driver genes." Objective. To further investigate this biomarker panel, the objective of the current study is to (1) validate the assay reproducibility for the 325 RNA biomarkers and (2) compare gene expression profiles side by side using two technology platforms. METHODS AND RESULTS: We have mapped the 325 RNA transcripts and in a custom NanoString nCounter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0. The experiments were conducted with 10 unique biological formalin-fixed paraffin-embedded (FFPE) breast tumor samples. Each site extracted RNA from four sections of 10-micron thick FFPE tissue over three different days by two different operators using an optimized standard operating procedure and quality control criteria. Samples were analyzed using mas5 in BioConductor and NanoStringNorm in R. Pearson correlation showed reproducibility between sites for all 60 samples with r = 0.995 for Affymetrix and r = 0.999 for NanoString. Correlation in multiple days and multiple users was for Affymetrix r = (0.962 - 0.999) and for NanoString r = (0.982 - 0.991). CONCLUSION: The 325 RNA biomarkers showed reproducibility in two technology platforms with moderate to high concordance. Future directions include performing clinical validation studies and generating rationale for patient selection in clinical trials using the technically validated assay.

Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples.

Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing. We examined RNA quality metrics conducted prior to RNA-sequencing in paired frozen and FFPE samples with varying levels of quality based on age in block and time in formalin. RNA quality was measured by the RNA integrity number (RIN), a modified RIN called the paraffin-embedded RNA metric, the percentage of RNA fragments >100-300 nucleotides in size (DV100-300), and 2 quantitative PCR-based methods. This information was correlated to sequencing read quality, mapping, and gene detection. Among fragmentation-based methods, DV and PCR-based metrics were more informative than RIN or paraffin-embedded RNA metric in determining sequencing success. Across low- and high-quality FFPE samples, a minimum of 80% of RNA fragments >100 nucleotides (DV100 > 80) provided the best indication of gene diversity and read counts upon sequencing. The PCR-based methods further showed quantitative reductions in amplifiable RNA of target genes related to sample age and time in formalin that inform input quantity of FFPE RNA for sequencing. These results should aid in screening and prioritizing archival FFPE samples for retrospective analyses of gene expression.

Selection of reference genes for quantitative real-time PCR normalisation in largemouth bass Micropterus salmoides fed on alternative diets.

The partial cDNA sequences of eight reference genes (actb, tuba1, gapdh58, gapdh59, eef1a1, RNA 18 s, pabpc1, ube2I) were cloned from largemouth bass Micropterus salmoides. The expression levels of these eight genes were compared in the various tissues (eye, spleen, kidney, gill, muscle, brain, liver, heart, gut and gonad) of M. salmoides fed on forage fish. The results showed that the candidate genes exhibited tissue-specific expression to various degrees and the stability ranking order was eef1a1 > tuba1 > RNA 18 s > pabpc1 > ube2I > actb > gapdh58 > gapdh59 among tissue types. Four candidate genes eef1a1, tuba1, RNA 18 s and actb were used to analyse the stability in liver tissues of largemouth bass between the forage-fish group and the formulated-feed group. The candidate genes also showed some changes in expression levels in the livers, while eef1a1 and tuba1 had the most stable expression in livers of fish fed on alternative diets within 10 candidates. So eef1a1 and tuba1 were recommended as optimal reference gene in quantitative real-time PCR analysis to normalise the expression levels of target genes in tissues and lives of the M. salmoides fed on alternative diets. In livers, the expression levels of gck normalised by eef1a1 and tuba1 showed the significant up-regulation in formulated feed group (P < 0.05) than those in forage-fish group. While sex difference has no significant effects on the expression levels of gck in both groups.

Establishing an RNA extraction method from a small number of Demodex mites for transcriptome sequencing.

Demodex is a type of parasitic mite which could cause serious dermatoses in 11 orders of mammals. However, due to the tiny body with thick chitin hard to be ruptured as well as the difficulty in obtaining a large number of mites, the quantity and quality of extracted RNA could hardly satisfied for transcriptome sequencing. This has hampered the research on functional genes and molecular pathogenesis of Demodex for a long time. To solve the problems above, the present study established a new RNA extraction method in combination Azanno method with liquid nitrogen grinding using 16 human and canine Demodex mite samples. The RNA quality detection results of Agilent 2100 Bioanalyzer showed that 8 of 16 RNA samples met the requirements for trace RNA-Seq, with RIN of 5.0-6.5 and RNA quantity of 1.1-16.0 ng. RNA quality was affected by grinding process and parasitic position of Demodex. Enough grinding number (≥2000) in moderate time (≤20 min) was significant for mites' complete rupture and RNA degradation prevention. D. brevis (100%, 3/3) parasitizing in human sebaceous glands had significantly higher RNA qualification rate than D. folliculorum (57.14%, 4/7) parasitizing in human hair follicles. Yet D. canis parasitizing in dog had lower RNA qualification rate (16.67%, 1/6) as mites were embedded in skin tissues and blood clots. It should be pointed out that microplate reader had defects with a lower RNA qualification rate of 6.25% (1/16) unmatched with 2100 Bioanalyzer, reminding that it could be only used as reference in RNA quality evaluation.

Three Methods to Purify Leukocytes and RNA Quality Assessment.

Leukocytes function as central effectors in innate immunity (such as phagocytosis) as well as adaptive immunity (e.g., antigen-dependent T cell activation), and serve as an important resource in the fields of translational medicine, precision medicine, and cell therapy. Isolation of leukocytes from whole blood is necessary for high-quality RNA and downstream research. This process is susceptible to the variability of many factors, such as blood collection, isolation reagents, and extraction methods. In this study, three methods were applied for leukocytes separation, followed by RNA extraction and quality testing to evaluate the methods. Results showed that leukocytes were purified using lymphocyte separation medium (LSM), optimized LSM method, or red blood cell lysis buffer (RBC lysis), and RNA quality met the basic requirements for downstream studies. Although considering the simplicity of the procedure and RNA quality from donated samples, the RBC lysis method should be recommended to biobanks for further research.

Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

Absolute Quantification of RNA Molecules Using Fluorescence Correlation Spectroscopy with Certified Reference Materials.

The accuracy and precision of quantification values of biomolecules, such as nucleic acids, are critical for the reliability of biomedical research and clinical examinations. To obtain an accurate quantitative value, it is necessary to use a measurement standard that has the same sequence and length as the target gene. The absence of an appropriate measurement standard leads to uncertain results. The development of a wide variety of different kinds of measurement standards, which have different sequences and lengths, is time-consuming and troublesome. We employed fluorescence correlation spectroscopy (FCS), which can be used to count the molecular number (absolute concentration) regardless of the molecular size and shape, without a standard curve. The confocal volume (i.e., the volume of excitation laser focus) of the FCS system was calibrated by measuring the primary standard of the fluorescent material. Furthermore, we investigated how to avoid artifacts originating from systematic aberrations or sample conditions. We validated the RNA concentration obtained from our FCS measurements using another primary standard RNA solution as a sample. Here, we describe an FCS calibration procedure with fluorescein solution standard reference material (SRM) 1932 as a primary standard and cross-validation of FCS values using RNA solutions certified reference material (CRM) 6204-a. The established method was applied to determine the concentrations of RNA samples that can be used as a laboratory working standards. The FCS method with a characterized SRM and CRM should serve as a universal method for absolute quantification of the number of biomolecules.

Modifications in routine protocol of RNA isolation can improve quality of RNA purified from adipocytes.

Adipose tissue is of interest in the context of its role in the pathogenesis of cardiovascular diseases. Modern experimental techniques require a well-purified RNA, but all the routine protocols for RNA extraction have a number of limitations in case of fatty tissues. Here we described a modified protocol for RNA extraction from human adipocytes based on routine column method. Suggested modifications optimized the sample preparation, lysis and washing lead to enhance RNA purity. We conclude that the current protocol for total RNA purification from adipocytes allows extracting a high-quality RNA devoid of fatty acids, organic solvents and salts contamination.

Long-Term Room Temperature Storage of Dry Ribonucleic Acid for Use in RNA-Seq Analysis.

RNA is an essential biological material for research in genomics and translational medicine. As such, its storage for biobanking is an important field of study. Traditionally, long-term storage in the cold (generally freezers or liquid nitrogen) is used to maintain high-quality (in terms of quantity and integrity) RNA. Room temperature (RT) preservation provides an alternative to the cold, which is plagued by serious problems (mainly cost and safety), for RNA long-term storage. In this study, we evaluated the performance of several RT storage procedures, including the RNAshell® from Imagene, where the RNA is dried and kept protected from the atmosphere, and the vacuum drying of RNA with additives such as the Imagene stabilization solution and a home-made trehalose solution. This evaluation was performed through accelerated (equivalent to 10 years for RNAshell) aging and real-time studies (4 years). To check RNA quality and integrity, we used RNA integrity number values and RNA-seq. Our study shows that isolation from atmosphere offers a superior protective effect for RNA storage compared with vacuum drying alone, and demonstrates that RNAshell permits satisfactory RNA quality for long-term RT storage. Thus, the RNA quality could meet the demand of downstream applications such as RNA-seq.

A Multivariate Evaluation of Factors Affecting the Quality of Freshly Frozen Tissue Specimens.

Well-prepared and preserved freshly frozen specimens are indispensable materials for clinical studies. To manage specimen quality and to understand the factors potentially affecting specimen quality during preservation processes, we analyzed the quality of RNA and genomic DNA of various tissues collected between 2002 and 2011 in Linkou Chang Gung Memorial Hospital, Taiwan. During this period, a total of 1059 freshly frozen specimens from eight major cancer categories were examined. It was found that preservation duration, organ origin, and tissue type could all influence the quality of RNA samples. The increased preservation period correlated with decreased RNA quality; the brain, breast, and stomach RNA specimens displayed faster degradation rates than those of other organs, and RNA specimens isolated from tumor tissues were apparently more stable than those of other tissues. These factors could all be used as quality predictors of RNA quality. In contrast, almost all analyses revealed that the genomic DNA samples had good quality, which was not influenced by the aforementioned factors. The results assisted us in determining preservation factors that affect specimen quality, which could provide evidence for improving processes of sample collection and preservation. Furthermore, the results are also useful for researchers to adopt as the evaluation criteria for choosing specimen collection and preservation strategies.

Influence of Freeze-Thaw Cycles on RNA Integrity of Gastrointestinal Cancer and Matched Adjacent Tissues.

BACKGROUND: Comparative analysis of RNA expression profiles between cancer and adjacent noncancerous tissues is an important part of cancer research. High-quality RNA is essential for consistent, reliable results, especially for identification of cancer biomarkers. However, the impact of freeze-thaw cycles on the quality of RNA both in gastrointestinal cancer and paired adjacent tissues is still unclear. AIM: To investigate the influence of freeze-thaw cycles on RNA integrity and overall histomorphology of gastrointestinal cancer and paired adjacent noncancerous tissues. METHODS: Gastrointestinal cancer and matched adjacent noncancerous tissues were frozen and thawed twice before extracting RNA. Total RNA in each sample was extracted with TRIzol reagents and the RNA integrity was assessed by RNA integrity number (RIN) on an Agilent Bioanalyzer. Light microscopy was then used to assess tissue composition and morphology. RESULTS: RIN values for all samples tended to decrease in correlation with the frequency of freeze-thawing. With an RIN cutoff value of 6, RNA extracted from pancreatic cancer tissues was not qualified after the first freeze-thaw cycle. Moreover, all RNA extracted from adjacent noncancerous tissues had nonqualifying RIN scores after the first freeze-thaw cycle, except for liver tissues. Microscopically, all samples displayed qualified tissue morphology regardless of freeze-thaw cycle frequency. CONCLUSION: Freeze-thawing affects the RNA integrity, but not the tissue morphology of gastrointestinal cancer and paired adjacent noncancerous tissues. Furthermore, the RNA extracted from adjacent noncancerous tissues is more easily degraded than that in cancer tissues.