Steroid receptors in the brain of female Japanese quail - a protocol for qPCR

Abstract

Based on a variety of studies investigating brain function in birds, it is now clear that birds possess a ‘social behavior network’, that is homologous to the social behavior network in mammals. Because the social behavior network contains steroid receptors, hormones play an important role in social behavior. The sensitivity to a certain hormone is determined by the expression of the receptor for this hormone. As a proxy for expression of a receptor itself, one can quantify amounts of RNA, which reflects gene expression. A very reliable method for determining the expression of genes, is the qPCR. For isolating RNA, different protocols were tested. A mixed protocol between isolation with trizol and isolation with the RNeasy Plus Microkit (50) gave the best results. After isolation of RNA, cDNA was prepared, as cDNA was necessary as starting material for the qPCR. For the qPCR, primers were tested for reference genes and for the different steroid receptors. The primers for reference genes gave better results than the primers for the steroid receptors, but no optimal results were reached yet. Amplification of cDNA started in the latest cycles of the qPCR, so it did not become clear if the right primers were used. For further investigation, I would suggest to continue using the mixed protocol for RNA isolation, and to keep investigating ways to start amplification of cDNA in earlier cycles of the qPCR, so it can be determined if the right primers are used.