RealTime PCR of microRNAs - (Sep/09/2006 )
I want to do RT PCT with microRNAs with the Applied Biosyst kit. Do you have any experences with microRNA RT??? experience with other kits or syst.???
What should I use us possitive and negative control??Thank you Marco
Are you using ABI miRNA assays? If so they include a RT primer to convert your specific mature miRNA of interest. You can find their protocol for the assays online.
ABI produces the best if not the only specific real-time miRNA assay.
I don't have a lot of exprience with random conversion of mature miRNA's but I don't think it is very efficient due to their short length (<30nt).
A lot of other companies use microarrays to profile miRNAs
I tend to use polyA tailed RT-PCR system, which you can prepare everything by yourselves.
Biotechniques. 2005 Oct;39(4):519-25.
GENES & DEVELOPMENT 20:2202-2207, 2006
If you want to do relative quant either,
Biotechniques. 2005 Oct;39(4):519-25.
(Plants)
Or
Cell Cycle 2006: 5 (17) 1951-1956
(Cell lines use Synth plant micro as + control)
With either of the above methods you can also do multiple micro’s with one RT'd sample (use spec primer for each) ABI can't do that 
What should I use us possitive and negative control??Thank you Marco
Do miRNAs have cap structure and poly A tail? There seems to have different views on this.
Both the above refs (see again below) refer to methods that add a polyA tail to the mature (18-22nt) microRNA which does not have either.
Quant PCR microRNA method orQuant PCR miRNA method
...but the pri-microRNA is reported to have cap n' tail.
In fact there are two strategies for analysis of miRNA by RT-PCR
One is based miRNA extension based, such as polyA tailing based RT-PCR (see above), adaptor ligation based RT-PCR (Exiqon); another is based on primer extension, such as ABI’s protocol(Nucleic Acids Res. 2005 Nov 27;33(20):e179.) and a similar assay described by paper (RNA. 2005 Nov;11(11):1737-44). Both strategies are proved work for mature miRNA. However, for analysis or pri/pre-miRNA, these method is not efficient, and you might use method described by paper in Nucleic Acids Res. 2004 Feb 25;32(4):e43.
One is based miRNA extension based, such as polyA tailing based RT-PCR (see above), adaptor ligation based RT-PCR (Exiqon); another is based on primer extension, such as ABI’s protocol(Nucleic Acids Res. 2005 Nov 27;33(20):e179.) and a similar assay described by paper (RNA. 2005 Nov;11(11):1737-44). Both strategies are proved work for mature miRNA. However, for analysis or pri/pre-miRNA, these method is not efficient, and you might use method described by paper in Nucleic Acids Res. 2004 Feb 25;32(4):e43.
Yes, that’s pretty much all that's available, however the polyA based methods (see above) RT all microRNAs in a sample this allows for "relative" quantitation. Variations in microRNA expression that are actually due to sample prep. can then be detected.
Neither of the commercially available kits mentioned above allows for relative quant.
could you please tell me whether I can use the RT-PCR method to see whether my overexpression of miRNA worked or not? many thanks. what reagent do I need? is it troublesome? what should be used for normalization?
As for endogenous control:
ABI offered several small RNA controls you could use. They would recommend you pick several and test for yourself which RNA is most unaffected in your samples, therefore use that as controls.
I used sno202, and it looks consistent to me.
Good luck!