Gene expression levels in small specimens from patients detected using oligonucleotide arrays.
Large-scale gene expression profiling using microarray technology is often limited by the amount of tissue or cells available. A number of RNA amplification protocols have been published to overcome this problem. However, additional amplification steps can result in both a 3' bias and poor reproducibility for low abundance transcripts. We performed microarray experiments using HG-U133A GeneChip arrays to ascertain whether less than the recommended amount of RNA can be used, thus avoiding additional amplification steps. In a titration experiment, 2-10 microg of total RNA from a single cryopreserved patient specimen was used to prepare biotinylated cRNA, and the recommended standard amount of 15 microg of each preparation was used for hybridization. Statistical analysis using box plots, correlation coefficients, MvA plots, and concordance percentages revealed almost identical levels of gene expression, independent of the amount of RNA used for target preparation. Most importantly, there was no statistically significant difference when the concordance percentages for low abundance genes were compared, demonstrating that as little as 2 microg of total RNA is sufficient to perform GeneChip analysis.
Hoffmann
Katrin
K
Division of Children's Leukaemia and Cancer Research, Telethon Institute for Child Health Research, P.O.Box 855, West Perth WA 6872, Australia. katrinh@ichr.uwa.edu.au
Firth
Martin J
MJ
Freitas
Joseph R
JR
de Klerk
Nicholas H
NH
Kees
Ursula R
UR
D002648:Child; D002675:Child, Preschool; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D007938:Leukemia / Q000235:genetics; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D018075:RNA, Complementary / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D018401:Sample Size; D012680:Sensitivity and Specificity
D018075:RNA, Complementary; D012334:RNA, Neoplasm