Selection of control genes for quantitative RT-PCR based on microarray data.
Use of internal reference gene(s) is necessary for adequate quantification of target gene expression by RT-PCR. Herein, we elaborated a strategy of control gene selection based on microarray data and illustrated it by analyzing endomyocardial biopsies with acute cardiac rejection and infection. Using order statistics and binomial distribution we evaluated the probability of finding low-varying genes by chance. For analysis, the microarray data were divided into two sample subsets. Among the first 10% of genes with the lowest standard deviations, we found 14 genes common to both subsets. After normalization using two selected genes, high correlation was observed between expression of target genes evaluated by microarray and RT-PCR, and in independent dataset by RT-PCR (r = 0.9, p < 0.001). In conclusion, we showed a simple and reliable strategy of selection and validation of control genes for RT-PCR from microarray data that can be easily applied for different experimental designs and tissues.
Shulzhenko
Natalia
N
Immunogenetics Division, Pediatrics Department, Universidade Federal de Sao Paulo--UNIFESP, Sao Paulo, SP, Brazil.
Yambartsev
Anatoly
A
Goncalves-Primo
Amador
A
Gerbase-DeLima
Maria
M
Morgun
Andrey
A
D000465:Algorithms; D014355:Chagas Disease / Q000235:genetics / Q000378:metabolism; D003627:Data Interpretation, Statistical; D020869:Gene Expression Profiling; D006084:Graft Rejection / Q000235:genetics / Q000378:metabolism; D016027:Heart Transplantation; D006801:Humans; D009206:Myocardium / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*