SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.
The small quantities of tissue available for most studies of human disease are a significant limitation for meaningful gene expression profiling. The Atlas Switch Mechanism At the 5' end of Reverse Transcript (SMART) probe amplification kit uses as little as 50 ng of total RNA to generate complex cDNA probes for DNA array and other analyses. However, the extent to which this attractive methodology maintains representation of relative gene expression has not been quantified. In this study, we demonstrate using real-time quantitative PCR analysis that the relative expression levels of a range of low- to high-abundance mRNAs are retained after SMART amplification independent of transcript abundance and full-length transcript, coding region and PCR product size. Using this technology, a mean amplification of 3800-fold was achieved in human liver samples, greatly enhancing the ability to perform replicate DNA array experiments. Probes generated with the SMART amplification method were used to detect increased expression of genes involved with inflammation, fibrosis, xenobiotic metabolism, immune function, oxidant stress and endothelium in liver from the baboon model of alcoholic liver disease.
Seth
Devanshi
D
Drug Health Services and A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital and Centenary Institute of Cancer Medicine and Cell Biology, Camperdown, NSW 2050, Australia.
Gorrell
Mark D
MD
McGuinness
Peter H
PH
Leo
Maria A
MA
Lieber
Charles S
CS
McCaughan
Geoffrey W
GW
Haber
Paul S
PS
D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000378:metabolism; D008108:Liver Diseases, Alcoholic / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D018076:DNA, Complementary; D012333:RNA, Messenger