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Lyophilized human cells stored at room temperature preserve multiple RNA species at excellent quality for RNA sequencing

ABSTRACT: Lyophilized human cells stored at room temperature preserve multiple RNA species at excellent quality for RNA sequencing

PROVIDER: PRJNA416340 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6235240.fastq.gz	Fastqsanger.gz
	SRR6235241.fastq.gz	Fastqsanger.gz
	SRR6235242.fastq.gz	Fastqsanger.gz
	SRR6235243.fastq.gz	Fastqsanger.gz
	SRR6235244.fastq.gz	Fastqsanger.gz

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Lyophilized human cells stored at room temperature preserve multiple RNA species at excellent quality for RNA sequencing

Project description:Purpose: We aimed to examine whether eukaryotic cell lyophilization and long-term storage would be feasible as an environmentally friendly and cheap alternative to cryogenic storage or the use of FFPE samples for large-scale RNA profiling studies. Brief method: On three different days three million GM12873 cells were taken up in 0.1 M trehalose, subjected to lyophilization (freeze-drying) and then stored for two weeks at room temperature in the presence of CaCl2 in a dark, tightly sealed box. Fresh paired control cells were pelleted and taken up in trizol without lyophilization. Total RNA was isolated using a modified trizol method, quantified and subjected to mRNA-Seq library preparation. Samples were sequenced using a NextSeq 500 instrument to 75 bp (single-end). Raw reads were aligned to the hg19 genomic build with TopHat 2.0.7 and gene expression quantification was performed using Cufflinks. Results: We found that lyophilized cells are highly similar in all inferred RNA-Seq quality metrics compared to their non-lyophilized controls and are useful sources of high-quality RNA for both small and large-scale studies. Conslusion: Lyophilization might be the preservation technique of choice in the future for studies aiming to involve large sample sets, such as clinical studies.

2018-08-13 | GSE106344 | GEO

Lyophilized human cells stored at room temperature preserve multiple RNA species at excellent quality for RNA sequencing.

Project description: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.

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Transcription profiling time series of human bone marrow samples stored at room temperature for up to 48 hours after aspiration

Project description:Impact of pre-analytical handling on Bone Marrow (BM) mRNA Gene Expression: We have investigated the impact of RNA extraction protocols and time delays (24 and 48h) between sample aspiration and RNA extraction on RNA quality and gene expression profiles. Intact RNA can be extracted from BM samples stored at room temperature for up to 48h after aspiration. However, storage of BM has dramatic effects on mRNA expression of individual transcripts.

2007-07-28 | E-GEOD-1347 | biostudies-arrayexpress

Analysis of metabolomics associated with quality differences between room-temperature- and low-temperature-stored litchi pulps.

Project description:Studies on how temperature affects the postharvest quality of litchi have focused mainly on pericarp browning but rarely on the metabolites in postharvest litchi pulp. In this study, the differences in respiration rates, total soluble solid content, and titratable acid content demonstrated that room and low temperatures have different effects on the quality of "Feizixiao" litchi pulp. UHPLC-ESI-QTOF-MS/MS analysis was performed to compare the differentially expressed metabolites (DEMs) in litchi pulp after 8 days of storage at room temperature (RT-8 d) with those in litchi pulp after 28 days of storage at low temperature (LT-28 d). Nineteen carbohydrates (phosphohexoses, sorbitol, and mannose), fifteen acids, seven amino acids, nine energy metabolites and nucleotides, and six aliphatic and secondary metabolites were identified as common DEMs in RT-8 d and LT-28 d pulps. These findings indicated active fructose and mannose metabolism and increased catabolism of nicotinate, nicotinamide, alanine, aspartate, and glutamate. Four carbohydrates (mainly phosphohexoses), five acids, ten amino acids, three aliphatic and secondary metabolites, and one hormone were identified as unique DEMs in RT-8 d pulp, the consumption of key metabolites in glycolysis and the tricarboxylic acid cycle, and accumulation of phenylalanine, tyrosine, and tryptophan. Active consumption of nucleotide metabolites and biosynthesis of aliphatics in LT-28 d pulp were indicated by unique DEMs (eleven carbohydrates, four acids, seven amino acids, seven energy metabolites and nucleotides, and six aliphatic and secondary metabolites). These results provided an unambiguous metabolic fingerprint, thereby revealing how room and low temperatures differentially influenced the quality of litchi pulp.

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Project description:When studying gene expression in microbe-animals symbioses collected in the field it is essential to quickly and efficiently preserve in situ symbiont and host gene expression patterns. One of the most commonly used sample preservation methods for samples targeted for proteomic analyses is flash freezing, however, liquid nitrogen or dry ice needed for flash freezing are often not available at remote field sites. We first tested if RNAlater allows to preserve proteins in animal-microbe symbioses as efficiently as flash freezing and without introducing issues with downstream processing (see PXD014591). Second, for the data in this PRIDE submission we tested if RNAlater preserves protein expression patterns over time at room temperature. We used the marine gutless oligochaete Olavius algarvensis as a test case. Olavius algarvensis lives in shallow water sediments off the coast of Elba, Italy. It has no digestive and excretory system and harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (Kleiner et al., 2012, PNAS 109(19):1173-82). For this dataset, we fixed a total of 33 worms and incubated them in RNAlater for up to 4 weeks. We then evaluated proteome preservation quality in terms of the number of identified proteins, abundances of individual proteins and potential biases against specific protein or taxonomic groups. Out of this 33 samples, eleven worms were incubated for 24 hours in RNAlater at 4°C (t0), while the other worms were incubated in RNAlater at room temperature (21-23°C) for additional 24 hours (t1, 6 worms), one week (t2, 8 worms), and four weeks (t3, 8 worms). We removed RNAlater from the worms after incubation and froze the samples at -80°C.

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