I recently knew about the PCR method for microRNA quantification using qPCR. Based on the papers (Balcells et al., 2011; Cirera et al., 2014) the method considers a single reverse transcription reaction using an extension primer (5′-ggtactagtttttttttttttttvn, v designates cytosine, guanine and adenine residues, n designates cytosine, guanine, adenine and thymine residues). I understand that I must use 12 primer (extension primers) in a concentration of 1 µM. The qPCR is performed using two primers (Forward and reverse) for each microRNA.
Can anyone share their experience using this method? I will appreciate any information about the accuracy and costs. Thank you.
I imagine you are talking about relative quantification. Why don't you simply use a miRNA TaqMan assay Kit from Applied Biosystems? Excellent products, high fidelity. You can quantify pri-miRNA, pre-miRNA or mature miRNA with this Kit. You can use a Primer Master Mix for quantification of up to 99 different miRNAs in one go.
Hi Lleretny,
I guess you're aiming the mature microRNAs here?
In that case theres a method called stem-loop PCR which makes you able to amplify the miRNA sequence by qRT-PCR (normal primers won't work due to the short size of mature miRNAs). There are online softwares which make u able to design your stem-loop primers.
cheers
Estimada Llerenty,
Yo vi la misma metodología y estoy por probarla. Estoy esperando que me lleguen los primers. De ahí podría contarte mi experiencia.
Saludos!
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