Currently, I'm working on a qPCR assay. I am using RT kits for my experiment . the setup that i had before for my assay is this :
For 1 Reaction: Add 2 RT Buffer + 4.5 H20 + 0.5 spike-ins +1 RT-Enzyme For 25 Reaction: Add 50 RT Buffer + 112.5 H20 + 12.5 spike-ins + 25 RT-Enzyme usually i make MM 10% more.
8 µl MM + 2 µl RNA to the reaction mini tube . i have 5 reaction tube so in total = 50 µl reaction. After RT i have 50 µl cDNA.
I could not detect any target in my qpcr, so i turned back to the handbook to re-setup my experiment .
in the handbook they mentioned these points:
- Use a template RNA volume equivalent to 16 µl original serum/plasma for each 20 µl reverse transcription reaction.
- Use a template RNA volume equivalent to 8 µl original serum/plasma for each 10 µl reverse transcription reaction. i have 2 quastions:
1:I do not know what is the best experiment set up according to my previous set up.is amount of cDNA enough?or how i optimize this assay?
2: is the RT calculation correct ?
Any recommendations or solutions would be appreciated.