Effect of multiple cycles of freeze–thawing on the RNA quality of lung cancer tissues

Abstract

RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.

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 .…”

“…RIN-based metrics have known confounders such as transcript level, and thus have been called into question as an appropriate quality metric 26 . For example, RIN failed to indicate a decrease in sample quality in lung cancer tissue samples that underwent ve freeze-thaw cycles 27 and, in statistical analyses, failed to correct for the effects of degradation 28 . Despite this, many studies rely on RIN to correct for and assess sample quality confounders 16,29,30 .…”

Multiple freeze-thaw cycles lead to a loss of consistency in poly(A)-enriched RNA sequencing

“…We found that while noise increases with both decreasing RIN and increasing freeze-thaw, RIN may be an insu cient indicator of quality for samples that have undergone two or more freeze-thaws. Given these results, RIN may not always be a good metric to quantify the difference between technical replicates that have undergone variable sample handling 17,[26][27][28] . We validate noise by con rming that it does not change with input RNA concentration, excepting outliers (Fig.…”

“…S1). Given previous indications that these metrics may not su ciently address transcript degradation 17,[26][27][28] , we instead measure the introduction of noise to samples (Fig S2-3). We de ne noise as the fraction of reads in a sample that are randomly counted, rather than mapping to a sample-speci c gene.…”

High‐throughput Preparation of DNA, RNA, and Protein from Cryopreserved Human iPSCs for Multi‐omics Analysis

“…Thermo Fisher Scientific instructions: Prepare a necessary amount of buffer by mixing QuBit DNA BR buffer solution with BR dye at a 200:1 ratio. RNA is particularly sensitive to degradation (Yu et al, 2017).…”