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miRNA real time qPCR problems

microRNA miRNA realt time PCR SYBR green

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#1 CamillaSteenJensen

CamillaSteenJensen

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Posted 25 November 2011 - 05:53 AM

Hi
I am currently trying to do miRNA profiling with real time qPCR as published by Chen et al., 2005, Nucleic acid.
I use total RNA isolated with trizol reagent from PBMC from donor blood. For the cDNA synthesis I use the stemloop method with a stemloop specific for all the miRNA's I want to studie, and the MuLV revers tranferase.
for the qPCR I use SYBR green and a miRNA specifik forward primer and a revers primer that attached to the stemloop.
My samples have a Ct of 20-24, but my problem is that my NTC come up around ct 30, and that my NRC come up around ct 21. So I can't seem to get a negativ samples.
Has anyone experienced this before?
I also tryed with synthetic tamplate = the sequence of the mature miRNA on DNA, and the same happens here. Dose anyone know if the app. Bioscinece MuLV revers tranferase can work on DNA as well as on RNA?
because that could be what i happing with the synthetic templates/miRNA's.
Thank for you help, in advanced...
Camilla





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