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Validation of the linear amplification of single bovine embryos

ABSTRACT: To generate expression profiles from single embryos, a protocol for linear amplification of RNA was developed on the basis of Baugh et al. (15). Linear amplification enabled production of sufficient amounts of aRNA for the microarray experiments and has been shown to reliably amplify the initial mRNA population (15, 18-20). Approximately 5 ng of total RNA can be isolated from a blastocyst-stage embryo (21); therefore, it was necessary to carry out multiple rounds of amplification. To test the reproducibility and fidelity of the amplification procedure, a validation experiment was conducted using RNA from the kidney tissue of a newborn calf. Two replicates of the bovine kidney RNA were amplified and hybridized to the microarray. Comparisons were made between aRNA (rounds 1, 2, and 3) and unamplified RNA. Keywords: cDNA microarray

ORGANISM(S): Bos taurus

PROVIDER: GSE3569 | GEO | 2005/11/30

SECONDARY ACCESSION(S): PRJNA93645

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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Validation of the linear amplification of single bovine embryos

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