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need help on microRNA real-time PCR - (Apr/07/2008 )

I'm now using microRNA Taqman® microRNA real-time PCR kit from Applied Biosystems to detect the expression level of microRNA in cultured cells. I used Trizol to isolate total RNA, and just follow the protocol provided by the kit using 10 ng total RNA, but I can not see the amplification using real-time PCR after 40 cycles as indicated by the protocol. Could anyone has experience on this help? Is there any other isolation methods or the purity of total RNA is a critical point? Thanks a lot in advance!

-lavenderyy-

i am not sure how the ABI system works, but there are a few others available. Invitrogen sells a first strand synthesis, and a NCode miRNA qPCR kit. I used it a few days ago.... didnt work for me, primers dimers, and some other problems perhaps, but you could try that.
Dont know what model system you use, but if you are using human, mice or I believe rats as well, EXIQON sells kits for miRNA qPCR with pre-designed primers for many different miRNA's. It seemed very extensive and their LNA technology is probably the best at this point for detection and quantification of miRNAs.
Hope this helps..

-Gallus gallus-

QUOTE (Gallus gallus @ Apr 14 2008, 07:45 AM)
i am not sure how the ABI system works, but there are a few others available. Invitrogen sells a first strand synthesis, and a NCode miRNA qPCR kit. I used it a few days ago.... didnt work for me, primers dimers, and some other problems perhaps, but you could try that.
Dont know what model system you use, but if you are using human, mice or I believe rats as well, EXIQON sells kits for miRNA qPCR with pre-designed primers for many different miRNA's. It seemed very extensive and their LNA technology is probably the best at this point for detection and quantification of miRNAs.
Hope this helps..




Thanks for the suggestions! I have tried out ABI qPCR kit, and the results turned out to be good:)

-lavenderyy-

QUOTE (lavenderyy @ Apr 17 2008, 04:04 PM)
QUOTE (Gallus gallus @ Apr 14 2008, 07:45 AM)
i am not sure how the ABI system works, but there are a few others available. Invitrogen sells a first strand synthesis, and a NCode miRNA qPCR kit. I used it a few days ago.... didnt work for me, primers dimers, and some other problems perhaps, but you could try that.
Dont know what model system you use, but if you are using human, mice or I believe rats as well, EXIQON sells kits for miRNA qPCR with pre-designed primers for many different miRNA's. It seemed very extensive and their LNA technology is probably the best at this point for detection and quantification of miRNAs.
Hope this helps..




Thanks for the suggestions! I have tried out ABI qPCR kit, and the results turned out to be good:)


the Taqman always works beautifully for me. the difference would be in RNA purity/ carry over of the phenol for the extraction protocol you used. the problem would re-appear in all assays you used , because phenol inhibits PCR reaction. The other problem might have been in reference gene you used.-- are you sure its expressed in your cell line?

-slave of science-

ABI's kit works great for me as well. Do you nanodrop your RNA after purifications? - if so you should look for a nice clean curve with ratios around 2 for your gamma readings. I use Ambion's MirVana kit to isolate as its columns preserve the small RNA fraction well - but Trizol should also work. What are you using as a positive control, if anything?

-Damsweet-

QUOTE (Damsweet @ May 4 2008, 07:06 PM)
ABI's kit works great for me as well. Do you nanodrop your RNA after purifications? - if so you should look for a nice clean curve with ratios around 2 for your gamma readings. I use Ambion's MirVana kit to isolate as its columns preserve the small RNA fraction well - but Trizol should also work. What are you using as a positive control, if anything?


i used ABI's kit for mirna qPCR in plants but it did not work. i made the same protocol as the manufacturer called, only i did not used amplitaq gold dna poly. can it be the reason?
i also tried to do the same experiment with sybr green mirna quantification protocol. but it did not work either. does anyone used this protocol before???
thanks
yasin

-yasin fatih-

QUOTE (yasin fatih @ May 7 2008, 03:15 PM)
QUOTE (Damsweet @ May 4 2008, 07:06 PM)
ABI's kit works great for me as well. Do you nanodrop your RNA after purifications? - if so you should look for a nice clean curve with ratios around 2 for your gamma readings. I use Ambion's MirVana kit to isolate as its columns preserve the small RNA fraction well - but Trizol should also work. What are you using as a positive control, if anything?


i used ABI's kit for mirna qPCR in plants but it did not work. i made the same protocol as the manufacturer called, only i did not used amplitaq gold dna poly. can it be the reason?
i also tried to do the same experiment with sybr green mirna quantification protocol. but it did not work either. does anyone used this protocol before???
thanks
yasin


Which miRs are you trying to probe for? I know this is probably a stupid question but did you use the Arapbidopsis assay kit? Just making sure as mammalian and plant microRNAs are rarely homologous. I've never used anything but the human kit so maybe the probes are just better designed or something? I'm sorry it's not working for you. What did you use instead of amplitaq gold? The human kit calls for Taqman Master Mix No AmpErase UNG.

-Damsweet-