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Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay
Published on: June 18, 2010
[Competitive RT-PCR assay for quantification of GPI-PLD gene expression]
Xiao-jie Zhang1, Chun-ping Lu, Jian-hua Tang
1Department of Biochemistry, Xiangya School of Medicine, Central South University, Changsha 410078, China.
Objective:
To establish a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to quantify the expression of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) gene.
Methods:
A competitive GPI-PLD cDNA fragment was constructed by site-directed mutagenesis by PCR method. The competitive cDNA fragment had the same primer binding sites as the target cDNA, but was 20 bp shorter in length. The competitive cDNA was transcripted into the competitive RNA in vitro as the internal standard. Then the competitive RNA was reversely transcripted and amplified together with total RNA extracted from K562 cells. The two amplified cDNA fragments could be distinguished by 10% polyacrylamide gel electrophoresis. The concentration of cellular GPI-PLD mRNA was derived from the ratio between the intensities of the bands corresponding to the amplified products.
Results:
A 198 bp GPI-PLD competitive RNA was constructed and prepared. The competitive RNA could compete well with the target RNA in the RT-PCR reaction. The expression of GPI-PLD gene in K562 cells was (440 +/- 20) copies/ng.
Conclusion:
A competitive RT-PCR assay for the quantification of GPI-PLD gene expression may be established successfully. This method is highly sensitive, specific, and accurate.
