Hi all current Honours student attempting to quantify a microRNA from leaf tissue. Carry out a standard Trizol extraction check RNA via nanodrop and agarose gel. This is then followed via stem loop RT and qPCR with SYBR green. Unfortunately am not getting ANY amplification. To remedy this I carried out the same experiment using borrowed RNA (alongside my own) borrowed stem loop primer for another microRNA (alongside my own) and used a known stem loop RT product for qPCR step (alongside borrowed and own samples). Still no amplification. Essentially I have tested the RNA and primers in one foul swoop and nothing. The big mystery the known stem loop RT product worked on a different qPCR run with different system (Taqman vs SYBR green power up master mix). Can anyone help me?
microRNA is challenging to quantify. I would recommend using TaqMan(®) MicroRNA assays.
These are some of the problems you can have and the solutions:
Inappropriate reaction conditions, Solution-Troubleshoot RT-PCR optimization.
Incorrect dye components chosen, Solution-Check dye component prior to data analysis.
Reaction component omitted, solution- Check that all the correct reagents were added.
Incorrect primer or probe sequence, solution- Resynthesize with appropriate sequence.
Degraded template or no template added, solution- Repeat with fresh template.
Reaction inhibitor present, solution- Repeat with purified template.
Hope this helps.
http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042996.pdf
http://www.ncbi.nlm.nih.gov/pubmed/20967604
I haven't done miRNA qPCR, but I have a couple thoughts. If you have a control that you know works in another system (Taqman) but not the one you're currently using (SYBER), there's a good chance there's something wrong with the way your SYBER assay is set up. Have you optimized your reaction conditions? If you're using the pre-loaded automatic settings on the qPCR cycler, or the same settings you used for Taqman, they might not work for your particular SYBER assay. You may need to try different annealing temperatures or amplification times. You might even be able to do some of this troubleshooting by normal RT-PCR, in a regular thermocycler with whatever polymerase you like to use. Then when you've gotten conditions close to worked out you can fine-tune them with the SYBER reaction mix in your qPCR cycler. It's probably also worth double-checking that your primers aren't in the reverse orientation if you haven't already.
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