Abstract
Biobanks operating at ambient temperatures would dramatically reduce the costs associated with standard cryogenic storage. In the present study, we used lyophilization to stabilize unfractionated human cells in a dried state at room temperature and tested the yield and integrity of the isolated RNA by microfluidic electrophoresis, RT-qPCR and RNA sequencing. RNA yields and integrity measures were not reduced for lyophilized cells (unstored, stored for two weeks or stored for two months) compared to their paired controls. The abundance of the selected mRNAs with various expression levels, as well as enhancer-associated RNAs and cancer biomarker long non-coding RNAs (MALAT1, GAS5 and TUG1), were not significantly different between the two groups as assessed by RT-qPCR. RNA sequencing data of three lyophilized samples stored for two weeks at room temperature revealed a high degree of similarity with their paired controls in terms of the RNA biotype distribution, cumulative gene diversity, gene body read coverage and per base mismatch rate. Among the 28 differentially expressed genes transcriptional regulators, as well as certain transcript properties suggestive of a residual active decay mechanism were enriched. Our study suggests that freeze-drying of human cells is a suitable alternative for the long-term stabilization of total RNA in whole human cells for routine diagnostics and high-throughput biomedical research.
Highlights
In the past two decades, large collections of biospecimens have been established worldwide in the form of tissue banks and have since become powerful engines of biomedical research
Quality and quantity of total RNA extracted from lyophilized cells after lyophilization As RNA molecules are inherently labile and sensitive to a number of factors, such as heat, oxidation, pH and especially cellular RNases, we first investigated the effect of our protocol and the freeze-drying cycle itself on the recovery and quality of total RNA extracted from human cells after lyophilization for six hours in 0.1 M trehalose
The RIN value might be an indicator of overall sample quality and is routinely assessed before sensitive gene expression analyses, such as microarrays and RNA sequencing (RNA-Seq), it has been argued that ribosomal RNA integrity may not reflect that of the mRNA fraction, partly due to structural differences between the two RNA classes [23,24,25]
Summary
In the past two decades, large collections of biospecimens have been established worldwide in the form of tissue banks and have since become powerful engines of biomedical research. Such biobanks represent invaluable sources of pathological samples for studies with various aims, such as identifying and validating biomarkers or uncovering cellular mechanisms underlying pathological conditions and drug resistance [1,2,3]. Tremendous effort has www.oncotarget.com been put into screening for biomarkers in a vast number of pathological conditions, utilizing large sample collections and high-throughput technologies, such as whole genome [5,6,7], exome [8,9,10] and RNA sequencing (RNA-Seq) [11, 12]. There is an increasing demand for datarich samples, especially when no clear future application is defined at the collection phase
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