Submitted to: American Society for Experimental Biology and Medicine Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 1/30/2004
Publication Date: 1/30/2004
Citation: Rosebrough, R.W., Poch, S.M., Russell, B.A., Richards, M.P. 2004.Using RT-PCR in a real time mode to determine expression of lipogenic genes in broilers [abstract]. FASEB Journal. 18:226.
Technical Abstract: Broiler chickens growing from 7 to 28 days of age were fed 12 or 30% protein diets and then switched to the diets containing the opposite level of protein. Birds were killed on days 28, 29, 30 and 31. In Experiment 2, birds were sampled, 0, 3, 6, 9 and 24 hr. following the switch in protein levels. Measurements taken included in vitro lipogenesis (IVL; Exp 1), malic enzyme activity (ME) the expression of the genes for ME, fatty acid synthase and acetyl coenzyme carboxylase. Gene expression was determined with a combined RT-PCR using SYBR green as a fluorescent probe monitored in a real time mode. SYBR Green binds all double stranded DNA, emitting a fluorescent signal on binding, and can be used for quantification of many targets. IVL and ME activity were inversely related to dietary protein levels (12 to 30%) and to acute changes in either level. Lipogenic gene expression was inversely related to protein level, whether fed on an acute or chronic basis. PCR amplification was monitored during its early phases of reaction and may be potentially more sensitive due to greater dynamic range (6-8 logs). The big disadvantage of combined system (RT-PCR) was that a large pool of cDNA was not produced as in a system that separates reverse transcription from PCR. Nonetheless, it appears that real time RT-PCR is an acceptable method of assaying gene expression in birds. In addition, further work will focus on primer sizes that might further optimize RT-PCR as an instrument for studying the regulation of avian lipid metabolism. Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out; however, that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.