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Simple ambient preservation of human tissues for RNA expression array analysis

ABSTRACT: We used quantitative RNA expression profiling on the Affymetrix U133 human expression array to validate quantitative expression results obtained with the tissue preservative RNALater against snap frozen and fresh tissues as a means of routine tissue collection and temporary storage. By using split samples from a homogenous tissue (uterine myometrium), and including duplicates within each processing group compared, we were able to undertake a formal ANOVA analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of test statistic values against which the observed results could be interpreted. Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. Keywords: parallel sample

ORGANISM(S): Homo sapiens

PROVIDER: GSE1296 | GEO | 2004/04/09

SECONDARY ACCESSION(S): PRJNA90217

REPOSITORIES: GEO

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Project description:Banking of high-quality placental tissue specimens will enable biomarker discovery and molecular studies on diseases involving placental dysfunction. Systematic studies aimed at developing feasible standardized methodology for placental collection for genomic analyses are lacking. To determine the acceptable timeframe for placental collection, we collected multiple samples from first and third trimester placentas at serial time points 0-120 minutes after delivery, simultaneously comparing the traditional snap-freeze technique to collection in commercial solutions designed to preserve RNA (RNAlaterTM, Ambion), and DNA (DNAgard®, Biomatrica). The performance of RNAlater for preserving DNA was also tested. Nucleic acid quality was assessed by determining the RNA integrity number (RIN) and genome-wide expression and DNA methylation microarray profiling. We found that samples collected in RNAlater had higher and more consistent RINs compared to snap frozen tissue, with similar RINs obtained for tissue collected in RNAlater as large (1 cm3) and small (~0.1 cm3) tissue pieces. RNAlater appeared to better stabilize the time zero gene expression pattern compared to snap freezing for first trimester placenta. Microarray DNA methylation analysis showed that overall the DNA methylation profiles remained quite stable over a two hour time period after removal of the placenta from the uterus, with the DNAgard condition being superior to both snap freezing and RNAlater. The collection of placental samples in RNAlater and DNAgard is simple, and eliminates the need for liquid nitrogen or a freezer on-site. Moreover, the quality of the nucleic acids and the resulting data from samples collected in these preservation solutions is actually higher than that from samples collected using the traditional snap-freeze method. Thus, this new approach to placental sample collection is both easier to implement in busy clinical environments and yields higher quality data. 72 samples

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Simple ambient preservation of human tissues for RNA expression array analysis

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