Hi, has anyone experience of miRNA qPCR panels, especially QIAgen miRCURY LNA miRNA PCR panel? I performed a qPCR run of 384 well plate (panel I) from SW480 cell line according to the procedure, but got very high Ct values. Spike-in control of RNA extraction, UniSp2, amplified at cycle 31.7, and Sp4 and Sp5 didn't amplify at all.
The total RNA yield was good (about 1 ug / ul). For the next run, I should probably add more RNA to cDNA synthesis and further to qPCR run, but how much?
I did a quick calculation:
Concentration differences between Sp2, Sp4 and Sp5 are 100-fold. Thus Sp4 should amplify 6.6 cycles later that Sp2, and Sp5 6.6. cycles later than Sp4. If I add cDNA template 10-fold more (increasing the amount from 20 ng to 200 ng), all Sp-controls should amplify 3.3 cycles earlier than in first run. Right? In my first run, Sp2 amplified on cycle 31.7. If I add 10-times more cDNA to the next reaction, Sp2 should amplify on cycle 28.4, Sp4 on cycle 35.0 and Sp5 as late as cycle 41.6, which is obviously too late.
What should I do?
Hi, yes I used the whole 20 ng, but that was for the whole plate, for 384 reactions. Thus the amount of total cDNA per one RT reaction is 52 pg.
Hello,
From my experience with cell lines, the yield of total RNA should be much higher.
" whole 20 ng, but that was for the whole plate, for 384 reactions. Thus the amount of total cDNA per one RT reaction is 52 pg"
I am not sure if i understood this correctly. Did you do 1 RT reaction (in a single tube) and from that 1 RT reaction you used 52 pg of DNA per well?
Also could you please look at the handbook for this kit https://www.qiagen.com/us/resources/resourcedetail?id=00a971e5-6dca-4223-8aaf-de7de9d3ed5f&lang=en
Could you please look at the figure with the header " Overview of issues and conclusions:" on page 14 and comment?
Also have you contacted a local sales rep/tech support? As i am from the bioinformatics area, i have contacted my local life science FAS here in Turkey but she didn't have experience with this kit. I will speak to other people locally and forward the case to EMEA tech support if necessary.
Hi Can,
Thank you for your comprehensive reply and sorry if I wasn't clear enough. Here I try to be more clear:
"From my experience with cell lines, the yield of total RNA should be much higher."
- I got much higher total yield of RNA from RNA extraction (approximately 1 ug/ul), but I used only 20 ng for RT reaction (as recommended in Handbook).
" I am not sure if i understood this correctly. Did you do 1 RT reaction (in a single tube) and from that 1 RT reaction you used 52 pg of DNA per well? "
- Yes, I did a single RT reaction from 20 ng of RNA and then used the whole reaction for mastermix that contained SyberGreen, buffer and this cDNA template. In miRNA qPCR panel the miRNA primers are placed in the wells, so I just pipetted 10 ng of mastexmix per well and then run the qPCR. (This is how the Handbook (https://www.qiagen.com/fi/resources/resourcedetail?id=f02862d7-29df-4466-a457-ebde78beb891&lang=en )recommends to do the qPCR reaction.) Thus 20 ng divided to 384 wells gives 52 pg per well. Right?
" Also could you please look at the handbook for this kithttps://www.qiagen.com/us/resources/resourcedetail?id=00a971e5-6dca-4223-8aaf-de7de9d3ed5f&lang=en
Could you please look at the figure with the header " Overview of issues and conclusions:" on page 14 and comment?"
- The Spike-ins in RNA isolation gave increased Cq, but Spike-ins of cDNA synthesis didn't. So the problem is in RNA isolation. However, as I mentioned, the total yield of RNA was good, but somehow the amount of miRNAs have reduced.
"Also have you contacted a local sales rep/tech support? As i am from the bioinformatics area, i have contacted my local life science FAS here in Turkey but she didn't have experience with this kit. I will speak to other people locally and forward the case to EMEA tech support if necessary."
- Yes, I have contacted Qiagen's (international) tech support, but the person that I was contacted with didn't have experience of RT or qPCR. I asked her to forward my query to someone who would have more experience but after that I haven't heard anything from them. :'DDDD I would be really really grateful if you could give some advises.
Taken together, my problem is that I'm not sure how much more RNA I should add to the RT reaction to get enough cDNA for the qPCR reaction. 20 ng that I used earlier is obviously too little. Would 200 ng be enough? That would give 520 pg of cDNA template per well.
Hello, Michael and his Finnish colleague will try to contact you regarding the matter. His email address is [email protected]
Hopefully it will be resolved soon. Thanks
-C.J
I just got a call from Mika (from Qiagen), and got some ideas how to continue. Thank you very much for your precious help!
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