Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling.
Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.
Nagy
Zsolt B
ZB
Laboratory of Functional Genomics, Biological Research Centre, Hungarian Academy of Sciences, P.O. Box 521, Szeged H-6701, Hungary.
Kelemen
János Z
JZ
Fehér
Liliána Z
LZ
Zvara
Agnes
A
Juhász
Kata
K
Puskás
László G
LG
D000818:Animals; D002460:Cell Line; D015342:DNA Probes; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D008070:Lipopolysaccharides; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D015342:DNA Probes; D008070:Lipopolysaccharides