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

Abstract

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.

Gene Expression Analysis of the Pre-Diabetic Pancreas to Identify Pathogenic Mechanisms and Biomarkers of Type 1 Diabetes

“…This is partly due to the lack of pancreas samples available for study, and the difficulty in extracting high-quality RNA from these tissues. High levels of endogenous RNAses, DNases and proteases present in the pancreas result in the rapid autolysis of tissues following tissue damage, and lead to varying levels of RNA degradation (16,73,74). This is frequently observed in tissues collected post-mortem, when extensive tissue processing times are unavoidable.…”

Comprehensive proteome and phosphoproteome profiling shows negligible influence of RNAlater on protein abundance and phosphorylation

“…In such cases, RNAlater may be the method of choice for tissue storage for subsequent molecular analysis of gene expression and mutation searches. RNAlater treatment was demonstrated to help obtain high-quality RNAs from human pancreas tissues [9], bacterial DNAs [10] and biological molecules [11]. A few studies have explored the effect of RNAlater on biomolecules such as DNA, RNA, and proteins.…”

Multiple Freeze-thaw Cycles Lead to a Loss of Consistency in Poly(A)-enriched RNA Sequencing

“…While some studies claim RIN can be used to account for degradation effects in RNA-Seq 16 , others suggest it does not su ciently capture the effects of degradation on sample quality 26,28 . When directly observing the effect of freeze-thaw on RIN, studies have found a negligible effect 12 or can only detect an effect after numerous cycles 27,50 .…”

“…Others account for "unwanted variance" due to technical, batch, or experimental variation. Yet, the in uence of sample processing, such as tissue lysis and processing time [12][13][14] , is not su ciently characterized such that it can be explicitly controlled. It is important to adequately characterize noise introduced to RNA-Seq measurements by sample processing steps to optimize sample quality, account for transcript degradation, and improve the accuracy and reproducibility of sequencing.…”