Reproducibility of alternative probe synthesis approaches for gene expression profiling with arrays.
Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 microg of purified poly(A)+ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 microg total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 microg total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard.
Centers for Disease Control and Prevention, United States Public Health Service, Department of Health and Human Services, Atlanta, Georgia 30333, USA.
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SD
Vernon
E R
ER
Unger
M
M
Rajeevan
I M
IM
Dimulescu
R
R
Nisenbaum
C E
CE
Campbell
D015342:DNA Probes / Q000138:chemical synthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000138:chemical synthesis / Q000235:genetics; D004076:Digoxigenin; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013223:Statistics as Topic; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D018076:DNA, Complementary; D012333:RNA, Messenger; D004076:Digoxigenin