1) I want to know how can i prepare standards for my qPCR. I will extract RNA from the brain followed by a DNAse treatment and the cDNA synthesis. I don't want to (if there are any) go for commercial available standards so kindly suggest how to prepare standards for qPCR and also please let me know if it is necessary to prepare standards?
2) What i know is i can make dilution series from my cDNA i.e. 1:1, 1:5, 1:25 and 1:125 and how do i decide which one to chose from the above dilution for further qPCR analysis?
3) I will use SYBR green master mix from thermo scientific. The protocol there says that my final vol. should be 20 ul. Can i reduce the volume or will it affect the qPCR?
4) Lastly, how can i decide the no. of cycles and the temperature or should i go for the builtin module? I am using stepone plus qPCR system.