Hi! It is my first time using TaqMan kits, so want to know something about Reverse Transcription process. I recently bought this Kit to extract microRNAs, to do the RT and to quantify on qPCR, and the handbook says that during the Reverse Transcription you can use a multiplex reaction, so my question is that about what does that means. Can I use several primers with a RT reaction? do I have to have another primers to be able to quatify another microRNA? If I use a single primer reaction, I can measure just one microRNA? and finally, I have an Universal primer that I have from another kit, can I use that to use some primers that I bought before?
Thanks for you help!
There are two kits from Life Tech: one is for just one or a few miRNAs with each RT reaction, another is Advanced miRNA assays for universal miRNA RT and a pre-amplification. We have tried both kits. If you have enough RNA material, I strongly recommend the first kit for single miRNA. For this first kit, you use one primer for RT to generate your miRNA-specific cDNA. Your miRNA assay will contain the other primer for the actual assay. I was told that you can multiplex your RT reaction with a few different primers. It is recommended to not use a universal primer from a different kit since you don't know the ligated 5' and 3'-oligo sequences to the miRNA at the RT steps. If you know these sequences, you will find out whether they will work together. Good luck.
I would not "mix and match" from another kit especially if you do not understand their chemistry. Can you send us which cDNA synthesis kit you are using?
The TaqMan assays use a unique stem-loop cDNA synthesis process. The forward primer will bind to miRNA sequence while the reverse will bind to the stem loop generated in the cDNA synthesis. See figure 1 of below link.
http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_042142.pdf
Multiplex means that a single PCR reaction can accomodate more than 1 fluorophore (i.e. SYBR), so that means within a single sample you can measure both gene of interest and the reference gene. Whereas with traditional SYBR green assays you had to use 1 reaction for gene of interest and 1 for the reference gene.
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