Dear researcher, could you please share me your experience?
My research is about mirRNA quantitative and qualitative study by PCR.
1. What is optimal miRNA concentration , for the further two step processes?
2. Which endogenous control do you consider the most suitable for Taqman mirna assays?
3. Before RT reaction do you perform preamlification reaction?
The set of reagents I am going to use for mirna quantitative and qualitative studies are:
1. TaqMan™ miRNA ABC Purification Kit - Human Panel A (Applied Biosystems™. ) – Cat. N 4473087
2. TaqMan® MicroRNA Reverse Transcription Kit Cat. N 4366596
3. TaqMan™ Universal PCR Master Mix- Cat. N 4304437
4. TaqMan® MicroRNA Assays- one tube assay
and
PCR machine- The Applied Biosystems® 7500 Real-Time PCR System.
Thanks in advance,
Hi，
You can find the answers for most of your questions in this doc：
https://assets.thermofisher.com/TFS-Assets/LSG/brochures/taqman-microrna-assay-faqs.pdf
Basically，10ng of RNA is enough for your Taqman miRNA Assay.
Hi, Zanda Bedinashvili
I agree with Xiaoguang, adding a suggestion: You can use a exogenous control to perform your analysis (like spike material), Thermo and Qiagen was available the cel-miR-39 (from C. elegans).
Greetings,
Maybe this section could help you (additional download the corresponding protokolls for kits used, to provide information on needed RNA conc. etc.)
Real-time PCR analyses -total RNA was isolated using universal RNA purification kit (Roboklon GmbH, Berlin, Germany).
First-strand cDNA synthesis was done using the TaqMan miRNA reverse transcription assay (Life Technologies GmbH, Darmstadt, Germany).
Real-time PCR was performed by using a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA), TaqMan miRNA expression assays for miR-29b, miR-497, and RNU48 (as control) all from Life Technologies GmbH, Darmstadt, Germany, following the manufacturer's protocol.
Conditions: 50°C, 2 min; 95°C, 20 s; 45 cycles 95°C, 3 s; 60°C, 30 s.
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