Comparison of standard exponential and linear techniques to amplify small cDNA samples for microarrays

BMC Genomics
Johan WadenbäckUlrika Egertsdotter

Abstract

The need to perform microarray experiments with small amounts of tissue has led to the development of several protocols for amplifying the target transcripts. The use of different amplification protocols could affect the comparability of microarray experiments. Here we compare expression data from Pinus taeda cDNA microarrays using transcripts amplified either exponentially by PCR or linearly by T7 transcription. The amplified transcripts vary significantly in estimated length, GC content and expression depending on amplification technique. Amplification by T7 RNA polymerase gives transcripts with a greater range of lengths, greater estimated mean length, and greater variation of expression levels, but lower average GC content, than those from PCR amplification. For genes with significantly higher expression after T7 transcription than after PCR, the transcripts were 27% longer and had about 2 percentage units lower GC content. The correlation of expression intensities between technical repeats was high for both methods (R2 = 0.98) whereas the correlation of expression intensities using the different methods was considerably lower (R2 = 0.52). Correlation of expression intensities between amplified and unamplified transcripts w...Continue Reading

References

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Citations

Dec 10, 2013·Analytical and Bioanalytical Chemistry·Ying ChenJinke Wang
Feb 7, 2008·International Journal of Plant Genomics·Robert C DayRichard C Macknight
Jul 22, 2009·BMC Genomics·Julie E LangChristopher M Haqq
Jan 30, 2008·BMC Genomics·Séverine A DegrelleIsabelle Hue
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May 9, 2007·Nanotechnology·Jason ReedJames K Gimzewski

Datasets Mentioned

BETA
GPL1880

Methods Mentioned

BETA
PCR
in vitro transcription
scrapings

Related Concepts

Bacteriophage T7 RNA polymerase
Transcriptase
Poly(A) Tail
Viral Proteins
Nested Polymerase Chain Reaction
Genes, Plant
Arabidopsis thaliana <plant>
Oligonucleotide Primers
cDNA Probes
Genome, Plant

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