I am trying to construct a standard plot for miR21 and miR 200 (Cq vs conc.) using synthetic oligos but even on using miRNA concentrations of upto 100pM I only begin to get any amplification after 40 cycles. I am using BioRad's iscript select cdna synthesis kit for the RT step and then ssofast evagreen kit for qpcr step. I'm also attaching one of the amplification curves and its corresponding melt curve (red is the NTC). Can anyone please point out where I could be going wrong?
Rachel GAD
thank you for your answer, the image seems to not have uploaded somehow, I'll do it now. Actually I was not using total RNA or any other biological sample as input, but synthetic mimetics for cDNA synthesis, that is ready-made oligonucleotides. Would I still need concentration in nano?
Rachel GAD
thank you, I'll try it out with ng of miRNA and let you know my results.
Hi Arti,
There several things about your experiments that "could be going wrong".
First, iScript Select cDNA Synthesis Kit protocol is for use with either oligo(dT) or random primers to reverse transcribe mRNA. This kit is not recommended to be used with miRNAs.
Second, there are several miR21 and miR 200 miRNA sequences: hsa-miR-21-3p, -21-5p, -200a-3p, 200a-5p, 200b-3p, -200b-5p, -200c-3p and-200c-5p. Make sure that your primers are specific for your target miRNA sequences.
Third, you did not identify what is the method to amplify miRNAs (obviously not TaqMan since Eva green is used for qPCR step) - therefore, it is impossible to see if you are using appropriate PCR primers (especially in combination with iScript Select cDNA Synthesis Kit).
I think every step from RNA to amplification is important and sensitive, you need to do practice more, more and more to set your own protocol. I am also agree with Rachel GAD RNA template concentration, you need to follow his suggestion, hopefully you will get amplification earlier.
Waqar Shafqat
Rachel GAD
, Waqar Shafqat I tried with 1 and 1.5µg of total RNA and also upto 100nM of synthetic oligos and still get the same results.
Sergei A Kazakov thank you for your answer. Actually the select kit also gives the option for gene specific primers and so I was using stem loop primers specific for the synthetic oligos I'm trying to quantify right now to plot a standard. Also for the qPCR step, I'm using forward and reverse primers specific for the CDNA and then evagreen intercalates in between causing fluorescence.
Rachel GAD
an update: I tried with someone else's primers for snU6 that I borrowed and I got Ct values of around 20 but the negative controls also give Ct values of 34 to 37. What do you think?
@Rachel, we are using miRNeasy kit for total RNA isolation and then using that for RT template.
@Rachel, actually getting a different kit would need approvals and would take some time so I was looking for solutions meanwhile. Also another lab here uses this kit for cDNA synthesis from miRNA.
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