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Whole Transcriptome Amplification for Gene Expression Profiling and Development of Molecular Archives

ABSTRACT: Type of experiment: This study used microarray experiments to evaluate the reproducibility and fidelity of Whole Transcriptome Amplification (WTA). Experimental factors: This study contains multiple categories of microarray experiments. Self-self hybridizations from WTA amplified total RNA were performed to evaluate the reproducibility of WTA. To evaluate the fidelity of WTA, the same total RNA, isolated from tissue samples or cell lines, was either directly used for microarray analysis or subjected to WTA amplification before hybridization. The amount of input total RNA for WTA amplification was varied for different hybridizations. To demonstrate the usefulness of WTA, benign epithelia, cancerous epithelia and stroma isolated by laser capture microdissection from frozen prostate tissue specimens were subjected to WTA and hybridized. To demonstrate the fidelity of WTA with degraded samples, total RNA from cell lines was artificially degraded before WTA and hybridization. Total RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues containing benign and cancerous prostate tissue were also subjected to WTA and hybridized. Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE2618 | GEO | 2006/01/02

SECONDARY ACCESSION(S): PRJNA92083

REPOSITORIES: GEO

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Project description:This SuperSeries is composed of the following subset Series: GSE3557: Effect of the amount of input total RNA on T7 amplification GSE3558: Effect of in vitro transcription time on the fidelity of T7-based RNA linear amplification GSE3559: Variation in cDNA microarray analysis of gene expression using unamplified poly(A)+ RNA GSE3560: Effects of template switching (TS) primer and cDNA cleanup columns on T7 based RNA linear amplification GSE3561: Effect of ligase on T7 based RNA linear amplification GSE3562: Effect of column cleanup on T7 based RNA linear amplification GSE3563: Correlation between expression levels of different tumors measured by poly(A)+RNA and aRNA Abstract: BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. Refer to individual Series

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Whole Transcriptome Amplification for Gene Expression Profiling and Development of Molecular Archives

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