Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance.
BACKGROUND
A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20-200 micro-grams total RNA or 0.5-2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material.


RESULTS
Using analysis of variance (ANOVA) and multiple hypothesis testing, we estimated the impact of amplification on the preservation of gene expression ratios. Both methods showed that the gene expression ratios were not completely preserved between amplified and non-amplified material. We also compared the expression ratios between the two cell lines for the amplified material with expression ratios between the two cell lines for the non-amplified material for each gene. With the aid of multiple t-testing with a false discovery rate of 5%, we found that 10% of the genes investigated showed significantly different expression ratios.


CONCLUSIONS
Although the ratios were not fully preserved, amplification may prove to be extremely useful with respect to characterizing low expressing genes.
Nygaard
Vigdis
V
Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. vigdisny@radium.uio.no
Løland
Anders
A
Holden
Marit
M
Langaas
Mette
M
Rue
Håvard
H
Liu
Fang
F
Myklebost
Ola
O
Fodstad
Øystein
Ø
Hovig
Eivind
E
Smith-Sørensen
Birgitte
B
D000704:Analysis of Variance; D018076:DNA, Complementary / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods* / Q000706:statistics & numerical data*; D006801:Humans; D016014:Linear Models; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000706:statistics & numerical data*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000706:statistics & numerical data*; D012333:RNA, Messenger / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm