The quantification of human factor IX gene expression in transfected mammalian liver cells, employing the real time qPCR and the ELISA techniques
karbala journal of pharmaceutical sciences,
Volume 5, Issue 7, Pages 218-226
AbstractReal-time qPCR and ELISA are very sensitive powerful techniques that were commonly and universally employed in the quantification of gene expression. The aim of this study has two parts; the first part is to quantify the level of the hFIX gene expression employing the real time qPCR for quantifying the newly transcribed mRNA. The second part is to detect the translated rFIX protein using the ELISA technique. In this study the mRNA levels present in mammalian liver cells (HepG2) that were transfected with hFIX gene has been successfully quantified by qPCR using hACTB as a housekeeping gene. Also the level of the translated rFIX protein has been observed utilizing the ELISA instrument. ∆Ct and ∆∆Ct for the transfected cells were -1 and -21 respectively, while the concentration of the rFIX in these cells was 5,495 ng/ml, which is more than 800 times increase when compared with the control cells. The validation of such methods, which are used for quantification of both the gene and the translated protein, is of significant importance. Furthermore the examination of the functionality of the target protein is even more important due to the challenges associated with these techniques.
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