Controls for real-time PCR... again - (Feb/25/2008 )
I've been thinking of quantifying some miRNAs by real-time RT-PCR using ABI microRNA Assays, but I have a huge question... how are you supposed to do normalisation (I thought of using an internal control) if you're using a sequence-specific RT primer?
I thought of doing separate RT reactions for each total RNA sample (one for miRNA and another for my housekeeping genes)... I know it's not a perfect control because RT is highly variable but I'd like to have more expert opinions
Sorry if this is a very stupid question, but I'm just starting to work with miRNAs and I've never done a real-time PCR before...
Thanks a lot!!!
I do what you said - separate RT reactions for each miRNA/control, and it works out pretty well. I use Rnu6b as houskeeping control - this is amplified in the same way as the miRNAs (i.e. hairpin RT primers) so I have more confidence that it is a valid approach. There is no other way for singleplex. I think that ABI recently released a multiplex system though, that you might want to check out (but when I checked, it was very expensive and there was no way to customize the components of each multiplexing kit).
Thank you so much for your answer... but now I have a different question.
I just want to do normalisation for relative quantification... would it be too wrong to quantify my housekeeping genes using regular RT and PCR primers and SYBR Green system while using the TaqMan Assays for miRNAs? I mean, there would be an error, but wouldn't it be similar for all samples?
Hmm, I'm not sure about that. I guess it could work, so long as you validate the efficiency of your SYBR primers (the TaqMan primers have an efficiency of >90%, I believe). I think the real question is whether the reviewers/editors would like it.
Thank you once again... I'm still going to think about it
I use snoRNA 202 as a housekeeping gene, applied biosystems have 10-15 housekeeping small RNAs which they recommend to use for normalization. it applies same design primers and probes as for the miRNA assays. I would not trust the SYBR green comparison to Taqman probes. you have to choose one assay either Taqman or SYBR, you cannot combine them.
hope that helps
you couldn't compare the taqman to real time.
have controls for the taqman, and controls for the real time.
controls are usually housekeeping genes. just keep in mind they should not change because of the treatment.
(on a side note, we have a guy who insists that people use at least 5 different genes as the controls (for 1 experiment) for their real time work.... i don't think so)
Thank you for your answers.
My problem is that ABI doesn't have controls for plants... but I guess I'll have to figure how to design primers and probes by myself