A1 Refereed original research article in a scientific journal
Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR
Authors: Hamalainen HK, Tubman JC, Vikman S, Kyrola T, Ylikoski E, Warrington JA, Lahesmaa R
Publisher: ACADEMIC PRESS INC
Publication year: 2001
Journal: Analytical Biochemistry
Journal name in source: ANALYTICAL BIOCHEMISTRY
Journal acronym: ANAL BIOCHEM
Volume: 299
Issue: 1
First page : 63
Last page: 70
Number of pages: 8
ISSN: 0003-2697
DOI: https://doi.org/10.1006/abio.2001.5369
Abstract
Real-time RT-PCR method was exploited to identify endogenous reference genes in differentiating human T helper cells. When using this technology in our experimental system, finding a set of genes whose mRNA expression levels would not change appeared to be very challenging. Our initial plan to use the expression level of GAPDH in normalizing the results failed, because the mRNA expression of GAPDH underwent significant changes during the cell culture. Additional studies on the transcription of several other classical housekeeping genes led to similar results. Our second approach was to use results from an extensive survey of gene expression done by oligonucleotide micorarrays and to select another panel of genes for testing. This resulted in the identification of three genes whose expression was relatively stable in our experimental system and, therefore, suitable as endogenous reference genes in these cells. The results indicate that the expression level of a constitutively expressed gene may change during the cell culture in vitro, which emphasizes again the importance of carefully validating endogenous control genes for comparative quantification. (C) 2001 Elsevier Science.
Real-time RT-PCR method was exploited to identify endogenous reference genes in differentiating human T helper cells. When using this technology in our experimental system, finding a set of genes whose mRNA expression levels would not change appeared to be very challenging. Our initial plan to use the expression level of GAPDH in normalizing the results failed, because the mRNA expression of GAPDH underwent significant changes during the cell culture. Additional studies on the transcription of several other classical housekeeping genes led to similar results. Our second approach was to use results from an extensive survey of gene expression done by oligonucleotide micorarrays and to select another panel of genes for testing. This resulted in the identification of three genes whose expression was relatively stable in our experimental system and, therefore, suitable as endogenous reference genes in these cells. The results indicate that the expression level of a constitutively expressed gene may change during the cell culture in vitro, which emphasizes again the importance of carefully validating endogenous control genes for comparative quantification. (C) 2001 Elsevier Science.
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