Preservation of RNA for functional analysis of separated alleles in yeast: comparison of snap-frozen and RNALater® solid tissue storage methods

Dekairelle, Anne-France; Vorst, Sébastien Van der; Tombal, Bertrand; Gala, Jean‐Luc

doi:10.1515/cclm.2007.281

Cited by 20 publications

(10 citation statements)

References 19 publications

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“…For RNA sequencing studies, the sources of technical variation are still being discovered, but can include many aspects of sample handling prior to actual measurement (McIntyre et al, 2011). Previous microarray studies have compared the sample handling procedures that were tested in our study and have found no difference downstream, particularly in differential gene expression patterns (Dekairelle, Vorst, Tombal, & Gala, 2007;Mutter et al, 2004). These studies, however, may not apply to the variance profile of RNA sequencing studies (Romero, Ruvinsky, & Gilad, 2012).…”

Section: Discussionmentioning

confidence: 87%

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

Passow

Kono

Stahl

et al. 2018

Molecular Ecology Resources

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RNA sequencing is a popular next‐generation sequencing technique for assaying genome‐wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field‐based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer‐term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome‐wide expression patterns in 30‐day‐old fry collected from the same brood at the same time point that were flash‐frozen in liquid nitrogen and stored at −80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash‐freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field‐based studies are substantial and should not be ignored.

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Section: Discussionmentioning

confidence: 87%

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

Passow

Kono

Stahl

et al. 2018

Molecular Ecology Resources

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“…Concerning an adequate storage condition for maintaining RNA integrity recent studies suggest the incubation of freshly obtained cartilage samples in the stabilization reagent RNAlater™ [11-13]. Here, we have to emphasise that the use of RNAlater™ results in a considerable curing process, thus impeding homogenization with a ball mill.…”

Section: Discussion and Future Developmentmentioning

confidence: 99%

Comparison of different methods for preparation and characterization of total RNA from cartilage samples to uncover osteoarthritis in vivo

et al. 2010

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BackgroundThe isolation of intact RNA can be very difficult when tissues are used that contain many RNAses or that are hard to homogenize, e.g. cartilage samples. Additionally, cartilaginous tissues are characterized by a low cellularity and an abundance of extracellular matrix (ECM) molecules. But given the growing interest in understanding pathogenesis of degenerative diseases, e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), studies have to consider expression pattern of cells in its natural environment.FindingsWe compared the current RNA isolation methods for the extraction of high-quality RNA of snap-frozen biopsies from limited amounts of hypocellular cartilaginous tissue. The focus of the study was to gather information about procedure-related differences in RNA quality and yield. Here, we describe two protocols, the phenol/chloroform-free filter-based method (RNAqueous™ kit) and the combined protocol (TRIzol®/RNeasy Mini™ kit), working in a reproducible and reliable manner.ConclusionsWe conclude that preparation, storage, homogenization, and quality control are altogether critical steps for in-depth analysis of differential gene expression, especially in hypocellular tissues with highly crosslinked ECM like cartilage.

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“…For now, most specimens use cold chains or are deepfrozen in the transportation and preservation process, but the emergence of novel storage reagents and technologies allows preservation of specimens at room temperature. For example, as an aqueous, non-toxic tissue storage reagent, RNAlater can rapidly permeate tissue to stabilize and protect cellular RNA in situ in unfrozen specimens, eliminate the need to immediately process tissue specimens or to freeze specimens in liquid nitrogen for later processing, this is convenient for simplified specimen handling and shipping (Dekairelle et al, 2007); there are well-documented high-quality nucleic acids preservation solutions such as GenTegra (IntegenX), RNAstable (Biomatrica) and RNAshell (Imagene), all of these commercial matrices could preserve DNA/RNA at room temperature for long-term and reduce energy costs (Mathay et al, 2012). All of these new types of preservation reagents and technologies, which are technologically feasible and economically reasonable both in quality and cost, provide new directions for specimen preservation at ambient temperature.…”

Section: (C) Novel Storage Technologiesmentioning

confidence: 99%

Chinese biobanks: present and future

et al. 2013

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As the demands of scientific research and application for specimens increase rapidly, biobanks in China have been springing up over the recent years. This paper summarizes Chinese biobanks through investigation and survey on operative, managerial, ethical conditions and challenges of biobanks. At present, hospitals and research institutes in China set up and operate most of the biobanks, collecting human specimens to support clinical and scientific research. With the development of bio-industry and arrival of the big data era, biobanks need not only collect and store human and non-human specimens but also to manage the big data associated with these specimens.

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Preservation of RNA for functional analysis of separated alleles in yeast: comparison of snap-frozen and RNALater® solid tissue storage methods

Abstract: Our results demonstrate that RNALater is a suitable and flexible alternative to snap freezing for FASAY analysis.

Cited by 20 publications

References 19 publications

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

Comparison of different methods for preparation and characterization of total RNA from cartilage samples to uncover osteoarthritis in vivo

Chinese biobanks: present and future

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