initial amount of cDNA - (May/06/2007 )
I'm new in Real time world and doing gene expression study, these are my assay;
I did RNA extraction with Trizol, use nanodrop to quantify RNA concentration and make cDNA with superscript kit (maximum 5ug/reactions in total 10ul, so I can put 8ul of RNA in reaction to get as much as cDNA) thus the amount of cDNA will vary depend on the initial RNA. I assume that I got 80-90% of cDNA from total RNA. In real time PCR plate, I made standard curve run along with tripicate cases and control samples. My questions are ...
1. I must run serial dilutions on every plate? can I use the standard curve from other plate?
2. How much cDNA of cases and control samples to put in reactions? Do I need to equallize the concentration of all cDNA samples before put in real time reaction?
3. How many house keeping genes to normalize 3 target genes? Can I use only one for those three genes? and can I use nuclear house keeping gene for mitochondrial target gene?
4. How many fold changes are significant?
any other recomend welcome
you must run serial dilutions on every plates.
we use 1 ug RNA to convert in cDNA. in reverse transcription reaction the reaction volume is 20 ul and i take 1-2 ul of sample from the pcr reaction tube and use it for real time pcr.
i cannot understand what do u mean by normalize 3 target genes but i use 2 housekeeping genes each time for many target genes
Many thanks for your answer
I did what you said, just feel that it's waste time and reagent and just got the same efficiency