Preserve Samples for RNA Expression Microarrays
RNAlater® Around the World
RNAlater® protects cellular RNA within specimens during sample collection and during shipping. Whether you are in the lab, in the OR, or out in the field, you can use this aqueous, non-toxic Tissue Collection:RNA Stabilization Solution from ambient temperatures down to -20°C.
Characterization of disease mechanisms using high throughput technologies often involves collaborations with a core facility for processing samples and data. Tissue procurement is a critical first step in the path to quantifying gene expression. Standardization of this step enables more accurate expression profile comparisons--both within a study and across published reports. The two studies described below demonstrate how RNAlater can protect RNA expression profiles within samples destined for array analyses without requiring specialized equipment (e.g. liquid nitrogen and associated containers) or handling (e.g. RNAlater is non-toxic and samples are stable at ambient temperatures).
Microarrays with Human Uterine Tissue
To expedite transfer of discoveries in genomic medicine to clinical care, Dr. George Mutter and colleagues conducted a comprehensive study to assess the impact of tissue sample source, tissue processing protocol, and random noise on microarray analyses [1]. Single uterine myometrium biopsies from randomly selected patients with benign uterine disease were subdivided into eight aliquots. Duplicate samples were either homogenized immediately, flash frozen in liquid nitrogen and stored at -80°C for 48 hours, or immersed in RNAlater at room temp for 24 or 72 hours.
In the expression profiles obtained from Affymetrix® HG-U133A arrays, samples from different patients had the greatest variability (measured by mixed model ANOVA, mean square error for genes with expression values >=100). Method of tissue collection and replicate samples from the same patient had the least variation, meaning microarray data obtained from routine repeat specimens from the same patient stored for 24 or 72 hours in RNAlater were not significantly different from those obtained when biopsies were fresh or flash frozen. In short, the variation in results observed between replicate samples from different tissue processing protocols was no greater than the variation between replicate samples from the same processing protocols. Thus, in this experiment, tissue could be stored in RNAlater at room temperature for up to three days without introducing unwanted variability to the gene expression analysis. These conclusions were further supported by data obtained from examining profiles of nine gene subgroups that consisted of multiple, functionally related gene classes.
Microarrays With Mouse Liver Tissue
Ambion scientists have also tested whether sample storage affects microarray expression profiling. Duplicate mouse liver samples were dissected and either frozen at -80°C for one week or stored in RNAlater at room temperature for 24 hours, 4°C for one week, or -20°C for one week. RNA was isolated (RiboPure™, Ambion), linearly amplified (MessageAmp™ II aRNA Kit, Ambion), and labeled with biotin-UTP before hybridization to an Affymetrix® MG 430A 2.0 array. The mean ± standard deviation for percent present calls for all samples was 59.4 ± 1.1, and the correlation between samples was excellent (range, R=0.988 to R=0.996). As a representative example, Figure 1 is a scatter plot summarizing the overall results when expression levels from samples stored in RNAlater for 24 hours were compared to that of samples frozen at -80°C.
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Figure 1. Scatter
Plot of Gene Expression Levels from Samples Stored
in RNAlater® vs
Samples Frozen at -80°C. Duplicate mouse
liver samples were dissected and either frozen at -80°C
for one week or stored in RNAlater at room temperature
for 24 hours, 4°C for one week, or -20°C
for one week. RNA was isolated (RiboPure™, Ambion),
and 1 µg total RNA was linearly amplified (MessageAmp™ II
aRNA Kit, Ambion). After a four hour in vitro transcription
to amplify and label samples with biotin-16-UTP, the
samples were hybridized to Affymetrix® MG
430A 2.0 arrays (22,690 genes). This scatter plot summarizes
the overall results when expression levels from samples
stored in RNAlater for 24 hours were compared
to that of samples frozen at -80°C. |
For more accurate gene expression profiling data, RNAlater can be used to stabilize samples during tissue collection and storage. The convenience and ease- of-use makes RNAlater ideal for preserving clinical and research samples that will be processed for microarray analyses.
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Scientific Contributors
Anton Zimmerman • Ambion, Inc.
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