Comparative evaluation of dexamethasone induced CYP3A and CYP2H1 gene expression by quantitative RT-PCR in Bantam, Bantamized white leghorn and white leghorn chicks
Kalia, Anil (2004) Comparative evaluation of dexamethasone 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.
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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. 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 -actin genes, respectively. - actin (house keeping gene) was used as an internal control for normalization of CYP3A 4 and CYP2H1 gene transcripts. Quantitative RT-PCR was done to quantify gene expression level of CYP3A and CYP2H1 genes. Four end points were selected for sample dropping at 26th, 31st, 36th and 41st cycles to perform Quantitative RT-PCR. The quantity of expressed genes was detected by Gene tool software using 1 kb DNA ladder having concentration of 7.1ng/0.5μl at 500 bp as reference. Relative expression ratio of CYP3A and CYP2H1 genes was calculated by Relative Expression Software Tool (REST). It was found that CYP3A is up regulated by a factor of 1.271 and 1.2 in Bantam and White Leghorn chicks, respectively and down regulated by a factor of 11.385 in Bantamized White Leghorn chicks. In Bantamized White Leghorn and White Leghorn chicks CYP2H1 gene was down regulated by factor 1.68 and 1.3 respectively, but up regulated by a factor of 1.126 in case of Bantam chicks. The PCR efficiency ranged from 1.4 to 1.9, 1.36 to 1.8 and 1.36 to 4.4 for CYP3A, CYP2H1 and -actin genes, respectively in Bantam, Bantamized White leghorn and White Leghorn chicks.
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