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Is RNA Amplification Necessary for Microarrays?

MC38) using mouse microarrays containing 2601 elements. In their experiments, all array hybridizations were repeated using reciprocal fluorescence to minimize the effects of labeling bias (i.e. each array was hybridized with with Cy3 labeled B16F10 RNA and Cy5 labeled MC38 RNA, and then a second identical array was hybridized with Cy5 labeled B16F10 RNA and Cy3 labeled MC38 RNA).

The following differences were observed between total RNA and amplified RNA samples:

(A) Correlation coefficients between reciprocal arrays were consistently higher with probes generated by a single or two rounds of amplification than with probes from unamplified RNA.

(B) Total RNA samples showed a higher correlation between duplicate forward or reciprocal arrays than between forward and reciprocal arrays. This was probably partially due to labeling bias. Total RNA also showed increased variability between duplicate arrays compared to amplified RNA. The authors speculate that data generated with total RNA (unamplified) may be more subject to the effects of background on the low-copy number transcripts than amplified RNA.

(C) The correlation between arrays using total RNA and amplified RNA was found to be similar to duplicate arrays probed with total RNA. This observation corroborates the finding that amplification does not significantly bias the gene expression data.

The authors further evaluated wh
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