AN IN VITRO RNA AMPLIFICATION STRATEGY ALLOWS CDNA MICROARRAY ANALYSIS OF SMALL AMOUNTS OF TISSUE

Abstract

189 We employ an in vitro mRNA amplification strategy based on the method originally described by Eberwine, et al., so that cDNA microarray analysis can be performed on small amounts of transplant biopsy tissue. Liver transplant core biopsies are obtained from patients at the time of transplant and at any time posttransplant when clinically indicated. mRNA is purified and converted to cDNA using an oligo dT primer containing a T7 RNA polymerase promoter sequence. An in vitro transcription reaction is then performed using T7 RNA polymerase, resulting in a thousand-fold linear amplification of the starting mRNA population. The amplified RNA is then labelled with fluorescently tagged nucleotides using reverse transcriptase and the resulting labelled probe is hybridized to cDNA microarrays containing as many as 10,000 human genes. The arrays are scanned with a laser confocal microscope which allows quantitation of mRNA levels. Several experiments comparing unamplified versus amplified RNAs from yeast and mammalian tissue culture cells have been performed. These show that the amplification strategy is highly reproducible and yields results that are representative of complex starting populations. As little as one nanogram of mRNA, or less than one tenth of a core biopsy, can be used. Amplification results in enough material to perform several hybridizations. Finally, we show that mRNA from clinically obtained specimens is successfully and reproducibly amplified using this method. Large scale differential gene expression experiments can now be performed on clinical samples of limited quantity. The RNA amplification strategy, in combination with cDNA microarray analysis, allows the in vivo exploration of gene expression changes during immunosuppression, rejection, infection, and malignancy on a near-genomic scale.