cdna synthesis - (Mar/27/2012 )
i just finished isolating my rna prior to reverse transcription and do qpcr after. i quantitated my total rna isolated and i just got confused if i should use same amount of my rna from 15 samples for reverse transcription. Should i calculate that each tube should have 2 ng per reaction tube? or should i just simply load 2 ul of each of my sample for reverse transcription?
if i am going to do qpcr, how much of the cdna product should i use and will that mean that all my 15 samples have equal amount of starting material for basis of comparison?
You should normalize your RNA according to grams (weight), not according to the volume.
We usually take 100 ng - 1 ug of RNA before synthesizing cDNA. Then we normally take 1- 2 ul of the cDNA reaction for our qPCR reaction, without measuring cDNA concentration. It works fine for us. It is difficult to measure cDNA concentration since it has RT enzyme, dNTP and buffer contamination. It's up to you however to measure it or not. you can also purify your cDNA before qPCR, but you will lose a lot of cDNA. If you want to measure cDNA concentration remember to adjust your spectrophotometer (for example Nnopdrop) to ssDNA.
If the template RNA is total RNA from cells make sure you use DNase to remove genomic DNA, because although you are using RNA extraction kits, it might still contain genomic DNA that might cause prroblem during qPCR as your primers might bind to gDNA and give wrong signal.