Universal Human Reference RNA for microarray expression
profiling
Pooled, High-Quality Reference RNA for Human Microarrays
Natalia Novoradovskaya • Terry Payette • Alexey
Novoradovsky • Jeff Braman
Stratagene
Nicki Chin • Alexander Pergamenschikov • Michael
Fero • David Botstein
Department of Genetics, Stanford University
Universal Human Reference
RNA for microarray gene expression profiling is a mixture of high-quality
total RNA isolated from 10 human cell lines. OD260/280
ratio of individual cell line RNA composing the reference
RNA is minimally 1.8. The RNA is intact and devoid of RNase activity as
judged by gel electrophoresis. The reference is efficiently labeled with
radiolabeled and fluorescent nucleotides and hybridizes to a majority
of microarray probe elements providing a consistent internal control and
allowing data comparison between different microarray experiments.
Reliable and precise microarray
gene expression profiling relies on comparison of hybridization efficiency
between experimental and reference RNA target samples1,2.
Differences in hybridization intensity between these RNA targets reflect
relative differences in gene expression levels. The easiest experiment
of this type to perform and analyze involves comparing the expression
profile of an experimental RNA sample resulting from exposure to some
perturbation relative to an unperturbed reference sample. The reference
sample in this experimental design is relatively easy to obtain. However,
for comparison of multiple experimental RNA samples, a common reference
composed of a collection of experimental RNA samples is required. The
importance of this common reference was emphasized by Eisen and Brown1
when they stated “This [referring to the reference] maintains the
essential internal-control aspect of two-color hybridization, but allows
inferred comparisons to be made among large numbers of samples without
requiring that every pairwise comparison be performed.” Accordingly,
examining a large collection of experimental RNA samples, within and between
gene expression experiments, requires a large quantity of reference RNA
composed of an equal mixture of all experimental RNA targets. The quantity
and complexity of this reference sample makes it extremely problematic
to obtain.
To address the necessity for large quantities of reference
RNA containing low and high abundance RNA species, Stratagene has developed
Universal Human Reference RNA for microarray gene expression profiling.
Total RNA Isolation
The reference RNA is composed
of total RNA isolated from cell lines representing different tissues using
a modified StrataPrep®
total RNA miniprep procedure.3
This method, based on RNA binding to glass fiber, produces high-quality
RNA free of protein, DNA and organic solvent contamination. Each of these
contaminants reduces RNA labeling efficiency (data not shown). Individual
cell line RNA included in the reference RNA possesses OD260/280
ratio no less than 1.8. Formaldehyde-agarose gel electrophoresis demonstrates
distinct 18S and 28S ribosomal RNA bands with no evidence of degradation
(Figure
1). Equal mass quantities of the highest quality total RNA from each
cell line are pooled to make the reference RNA.
Optimized Hybridization Coverage of Microarrays
Fig. 1
Cell lines for reference
RNA are chosen based on several important criteria. The foremost is that
cDNA produced from the reference RNA hybridizes to as many microarray
probe elements as possible. The reference RNA cell lines providing these
consistent and reproducible RNA populations include testis, brain, liver,
skin, breast, cervix, T and B- cells, macrophages, and lipocytes.
When considering optimized
coverage on a microarray, a pool of RNA from too many cell lines will
result in dilution of low-abundance genes represented in each individual
cell line. Therefore, optimized the reference RNA was prepared from the
fewest number of cell lines expressing the largest number of genes hybridizing
to microarrays (data not shown). To determine the percentage of array
probes hybridizing to individual cell line RNA and the reference RNA,
total RNA from these samples were reverse transcribed with [a33-P]
dTTP and radiolabeled cDNA was hybridized to Stratagene’s GeneConnection™
Discovery microarray.4
Microarrays were visualized using a phosphorimager (Packard Bioscience)
and analyzed using OptiQuant Image Analysis Software. Signal distribution
among more than 4000 spots on the microarray demonstrates that cDNA derived
from HURR hybridizes to a higher percentage of array probes than cDNA
from any individual cell line (Figure
2).
Fig. 2
Further evidence demonstrating
this point was obtained by reverse transcribing a single cell line RNA
and the reference with Cy5-dUTP and Cy3-dUTP, respectively, followed by
hybridization to a 24,000-spot human microarray from Stanford University.
The general procedure for this experiment is shown in Figure
3. The array was scanned using an Axon scanner and analyzed using
GenePix Pro 3.0 software. Figure
4 shows a portion of the microarray image and the ratios of red to
green signals were used to compare gene expression profiles in different
cell lines. Once again, the reference RNA hybridized to a larger number
of array probes than the individual cell line RNA.
Fig.3
Fig. 4
Conclusions
Universal Human Reference RNA is a unique blend of high-quality
total RNA from 10 carefully chosen human cell lines. Using the same reference
RNA in different microarray experiments provides a common denominator
for accurate and reproducible comparison of gene expression data. In addition,
use of the same reference RNA among different research groups allows inter-laboratory
comparisons as well. We recommend using Universal Human Reference RNA
as a reference sample in any multicolor hybridization experiments using
human cDNA microarrays.
REFERENCES
1. Eisen, M.B. and Brown, O. (1999) Methods in Enzymology
303: 179-2.
2. Ross, D.T., et al. (2000) Nature Genetics (2000) 24: 227-235.
3. Dolter, K. et al. (2000) Strategies 13: 12-14.
4. Garrison, D. et al. (2000) Strategies 13: 4-5.
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