Reliability of t7-based mRNA linear amplification validated by gene expression analysis of human kidney cells using cDNA microarrays.
Genome wide gene expression analysis by cDNA microarrays is often limited by minute amounts of starting RNA. We therefore tested an optimized linear RNA amplification protocol using the RiboAmp amplification kit in the setting of cDNA microarrays. We isolated mRNA from a human kidney cell line (HK-2; ATCC) and from Universal Human Reference RNA (STR; Stratagene). After performing one and two rounds of linear RNA amplification, respectively, the amplified RNAs were co-hybridized to cDNA microarrays. Linearity and reproducibility of the individual experiments were then assessed by calculating the Pearson correlation. The intra-amplification consistency showed a correlation of 0.968 for the first round, 0.907 for the second round and 0.912 for two successive rounds of amplification. If the first round was compared to unamplified material, r was 0.925. The second round amplification yielded a correlation of 0.897 if compared to unamplified mRNA. Two rounds of amplification starting from 200 pg of mRNA compared to unamplified material resulted in a correlation of 0.868. These results indicate that linear amplification using RiboAmp kit yields amplified RNA with a high degree of linearity and reproducibility.
Department of Nephrology, University Hospital of Innsbruck, Innsbruck, Austria. michael.rudnicki@uibk.ac.at
Michael
M
Rudnicki
Susanne
S
Eder
Gabriele
G
Schratzberger
Bernd
B
Mayer
Timothy W
TW
Meyer
Martin
M
Tonko
Gert
G
Mayer
D002460:Cell Line; D003199:Computer Systems; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*; D014764:Viral Proteins
D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases