I’ve been performing, so far, my qPCRs reactions in 50 uL of final volume; however, at doing in this way, I have spent a lot of material. In this sense, I would like to know if I could perform my qPCRs reactions using only half of that volume ( 25uL) without it interfering in my analyzes once I already started the analyzes of some replicates of both treated and control groups into my donors, and I can not have wide variatons in the genic expression among the replicates and the donors, otherwise I will lost my replicates given that the data will not be realiable.
I should be fine with 25ul also since your are using housekeeping gene and controls with the same volume
Try 50, 25 and 10 with same primers and conditions (control vs treated), and you will see if the result is the same (at least the trend).
Good luck
Hi Amando
to avoid lot of loss, you can adjust your PCR to 10ul final, but just be sure all yours RNA samples will have the right concentration and qualities (RIN).
fred
Hi Amando
Greetings
i working in qRT-PCR usually and i used all conditions in the same point
first, measurement of RNA or DNA concentration. second, qRT-PCR reactions including: reference gene (any primer from housekeeping gene such as: beta-actine, 18sRNA,.......), sample control and finally treatment samples, then for each sample was used 3 replicates and always the reaction volume is 25uL and give me good result
Good luck
- Badges
- Science topic