miRNA array data and QPCR data correlation - (Apr/17/2008 )
I'm using QPCR to confirm miRNA array data at present, but unfortunately, the results do not corelate with each other. Has anyone here has experience to do with these two methodologies? Thanks a lot!
When you say they don't correlate, do you mean that you see opposites (e.g. upregulation in array and downregulation in PCR)? Or are the fold changes slightly different? I think it would be unlikely to have identical fold changes in both, but they should be similar.
i made same experiments and the results were not very promising. i am repeating them know. so in a few weeks i can tell you better...
Hi,
Regulation of miRNAs found by Q-PCR does not necessarily correspond well to arraydata for all miRNAs. We've seen it for miR-9.
All the Q-PCR assay-design I've come across measure both mature and precursor miRNAs. So, if you for example have massive amounts of precursor miRNA, which is not regulated, but your miRNA is, then Q-PCR may not display the same regulation as your array did.
Do a northern blot - that will show you both the regulation of precursor and mature miRNA species.
Louise
Data of Array and qPCR for miRNA might not identical, The reason might relate to different specificity between PCR and array:
It is well known that hybridization based technique, especially using short oligo probe could be sensitive to mismatches in the middle of sequence between probe and target, but less sensitive to mismatch at the terminal of probe or target; while qPCR, is very sensitive to mismatch between 3’ terminal of primer and target sequence. This phenomena would be more obvious for RT-PCR designed for miRNA. In most cases, members in the same miRNA family are usually found to be different at 3’ terminal, and such difference might not easy to be discriminated using hybridization based technique, including array.
Regulation of miRNAs found by Q-PCR does not necessarily correspond well to arraydata for all miRNAs. We've seen it for miR-9.
All the Q-PCR assay-design I've come across measure both mature and precursor miRNAs. So, if you for example have massive amounts of precursor miRNA, which is not regulated, but your miRNA is, then Q-PCR may not display the same regulation as your array did.
Do a northern blot - that will show you both the regulation of precursor and mature miRNA species.
Louise
TaqMan based miRNA assays from ABI only amplify the mature sequence. They say that they can also distinguish between miRNAs with single base differences. But on this latter point I'm not so sure.
If precursor can be detected by nothern hybridcation, then array signal might still contain precursor's signal as well, unless you use fractioned small sized RNA as template.
