Comparative evaluation of phenobarbital induced CYP3A and CYP2H1 gene expression by quantitative RT-PCR in Bantam, Bantamized white leghorn and white leghorn chicks
Goriya, Harshad (2004) Comparative evaluation of phenobarbital induced CYP3A and CYP2H1 gene expression by quantitative RT-PCR in Bantam, Bantamized white leghorn and white leghorn chicks. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, IDNIA.
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The present work was planed to study induction of CYP3A and CYP2H1 genes by Reverse Transcription polymerase chain reaction (RT-PCR) and Quantitative RT-PCR in Bantam, Bantamized White leghorn and White leghorn chicks. Total RNA was extracted from the liver tissue samples using Tri Reagent® based method. The quality of RNA extracted ranges from 1.7 to 2.0 OD ratios and quantity of RNA was also good obtained. First strand cDNA was synthesis one step RT–PCR Kit. The PCR was performed and the product was subjected to agarose gel electrophoresis which yielded targeted amplification of 1107 bp, 1567 bp and 486 bp amplicon for CYP3A, CYP2H1 and β-actin genes, respectively. β-actin (house keeping gene) was used as an internal control for normalization of CYP3A and CYP2H1 gene transcripts. Quantitative RT-PCR was done to analyze the quantitative gene expression level of CYP3A and CYP2H1 genes. Four end points were selected for sample dropping at 26th, 31st, 36th and 41st cycles by using Relative Expression Software Tool (REST), it was found that CYP3A is up regulated by factor of 1.339, 14.507 and 1.004 in Bantam, Bantamized White Leghorn and White Leghorn chicks, respectively. In Bantam and Bantamized White Leghorn birds CYP2H1 gene was up regulated by factor 1.503 and 80.871 respectively, but down regulated by a factor of 1.965 in case of White Leghorn chicks. PCR efficiency was judged by Ling PCR software. The PCR efficiency ranged from 1.3 to 1.7, 0.86 to 1.7 and 0.91 to 1.58 for CYP3A, CYP2H1 and β-actin, respectively in Bantam, Bantamized White leghorn and White Leghorn.
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