I am currently working on tea miRNAs. For RT-qPCR analysis i need to use the U6 as control. I have worked out the primers for miRNAs, and also have forward and reverse primer for all. But how can i design the RT primer for U6snRNA?
Ordering via ABM is one option.
e.g. see https://www.abmgood.com/U6%20snRNA-Primers-Mouse-MPM00002.html
-If you are looking for miRNA primer including endogenous control, then prefer pre-made primer from companies. miRNA primer have universal sequence, upon which company synthesize with little chemical modification to increase stability and to enhance specificity.
-Qiagen U6 primers, which come is pre-mix content (FP and RP mixed in one vial, just dissolve, it ready to use)
-Prefer to use 2-3 reference endogenous miRNA control based on cell type and condition. **Never rely on only one control**
Hi, Debasish B Krishnatreya
As others told you, I'd say you to buy from one of the major companies around the corner on miRNA technology. It's that much expensive. But, If you really need to do it, check a few softwares like this one: Article A tool for design of primers for microRNA-specific quantitat...
About controls, really, don't work without a few ones. If you're aware about the miqe Guidelines for qPCR and qRT-PCR it will be clear to you. If not, give it a chance!
The best thing would be to check literature on your specific field and then try 4 - 10 miRNAs. As much as possible for your budget.
Take a look at this example:
http://cgp.iiarjournals.org/content/13/1/63.full
QIAGEN's list of controls:
https://www.qiagen.com/us/products/discovery-and-translational-research/pcr-qpcr/qpcr-assays-and-instruments/mirna-qpcr-assay-and-panels/miscript-pcr-controls/#orderinginformation
Anyway, data about U6 can be seen here:
https://systembio.com/wp-content/uploads/SBI_QM_control_assays-1.pdf
Last, remind yourself some published works may give you wrong info ... Do your best evaluation and contact whoever you think may help you. Check a chance to contact the leaders in this field (qPCR, qRT-PCR) such as Drs. Vandesompele, Kubista, etc. They are probably around here.
Hope it helps you.
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