Comparative Evaluation of Phenobarbital Induced CYP3A and CYP2H1 Gene Expression by Quantitative RT - PCR in Bantam, Bantamized White Leghorn and White Leghorn Chicks
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Date
2004
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AAU, Anand
Abstract
The present work was planned to study induction of CYP3A and CYP2H1 genes by Reverse Transcriptase polymerase chain reaction (RT-PCR) and Quantitative RT-PCR in Bantam, Bantamized White leghorn and White leghorn chicks. Out of 18 chicks of Bantam, Bantamized White leghorn and White leghorn, 3 from each group were treated intraperitoneal with phenobarbital at the dose rate of 12mg/I00gm body weight and control group were treated with same volume of 0.9% normal saline. After 24 hrs of medication, they were sacrificed and liver samples were collected from each bird. Total RNA was extracted from the liver tissue samples using Tri Reagent® based method. The quantity of extracted RNA was assessed spectrophotometrically at 260/280nm, and it ranged from 1.7 to 2.0 OD suggesting good quantity of RNA extraction. The quality of extracted RNA was checked by 1% formaldehyde agarose gel electrophoresis and it showed bands at 28s, 18s and 5s rRNA subunits suggesting good integrity of RNA. First strand cDNA was synthesized using 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 p-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 quantify gene expression level of CYP3A and CYP2HI genes. Four end points were selected for sampling at 26th, 31st 36rd and 41st cycles to perform Quantitative RT-PCR. The quantity of expressed genes was detected by Gene tool software using Ikb DNA ladder having concentration of 7.1 ng/0.5 μl at 500bp as reference. Relative expression ratio of CYP3A and CYP2H1 genes was calculated by Relative Expression Software Tool (REST), ft 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 chicks CYP2HI gene was up regulated by factor 1.503 and 80.871 respectively, but down regulated by a factor of 1.965 in 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 p-actin, respectively in Bantam, Bantamized White leghorn and White Leghorn chicks.
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VETERINARY PHARMACOLOGY, A Study