I am starting with microRNA isolation from serum, and the Ct values for miR16 I get using assays from Applied Biosystems for mature microRNA are around cycle 30.
The Ct values depends on different factors, the extraction procedure, the amount of serum, the amount of RNA used for RT and the amount of template in your qPCR reaction. It is not possible to generalize. With a miR-16 at a Ct of 30 I expect you will miss many less intense signals.
I agree with Andrea, Ct value can vary. If it is coming too low, then dilute the template, and if too high, use more template. There are other factors involved also, but template amount is one major factor which will affect Ct values.
Thanks for all your answers, I suspect that my problem is during RNA isolation, I am using 250 microL from serum, 2 microL o Glycogen and 1 mL of tripure reagent and re-extraction with 750 microL of tripure. I've done 10 and 50 ng RNA (nanodrop measurments) RT reactions with the ABI protocol, and the cDNA input in PCR 50 microL reaction is 10 microL. I also used PBMCs as control and the Ct values for miR-16 are around 15. Two questions:
Should I try miRNeasy Serum/Plasma Kit or Trizol LS?
Do 260/280 and 260/230 ratio assessed with Nanodrop need to >1.6 and >1 respectively?
The quantification with Nanodrop or Agilent can not be accurate for miRNA extracted from serum. The two techniques are not so sensitiv unfortunately. The only quantification technique still remains the qPCR.
I agree with Andrea. I was never able to quantify anything coming serum with Nanodrop. I worked routinely with 300 ul of human serum for extraction using a serial protocol with Trizol LS and Qiagen colums for cleanup. The only way to see something is to use Bioanalyzer pico-chips. I am attaching a typical profile of a RNA extracted from serum as you should see in the Bioanalyzer pico-chips.
About the Ct of the mIR16, I think that your value is pretty accurate. I am obtaining values around 28-32 using the Exiqon LNA primers. The values for Ct using the ABI protocol sound to me very optimistic for such a small amount of starting material.
Thanks Francisco of the PDF. Actually you recovered a great amount from 300 µl of serum! The Agilent profile is very close to our profile, exept that the amount is lower (100 pg/µl) for 1 ml of serum. Maybe Trizol LS is more effective than Norgen kit. I ordered Trizol LS and I will made some comparisons.
The only thing that worries me is the presence of degradation products: I do not know if this may depend on the extraction procedure, or if is it normal considered that you are using serum. Trizol let you recover more RNA (compared to other kits), but maybe also more degradation products...have you tried to quantify only the small RNA peak near the lower marker? Which is the amount in pg/µl of only the first peak? The baseline should be adjusted a little bit, but I expect that the amount should be much lower.
Let me know!
Bye
Andrea
Andrea
Degradation products are common in such a starting samples. I did not quantify the small peak close to the marker... this should be the peak of the small RNAs. However I am not too sure that the smear is only constituted by degradation products. It could be also other RNA species also present in plasma. The original paper describing circulating miRNAs also described mRNAs in circulation.
Trizol LS works OK in my hands but we never used it alone for serum. We used the Trizol LS without precipitation and then we run a Qiagen column for RNA purification.
This protocol is described by the people from Exiqon. I do not remember if I already sent you the protocol. It is attached to the message. By the way we never used the MS2 carrier... and if works...
Good luck my friend.
Hi Andrea, I think one of your problems come from the volume of starting materials. I have used the Plasma/Serm Circulating RNA Norgen kit (http://www.norgenbiotek.com/display-product.php?ID=465) for the purifcation of small RNA (
Hi Moreau, I also tried the kit you mention and I found it very good. I will also try Trizol LS and I will made some comparisons. I will also try the modification of Francisco although with other columns. I will let you know.
Thanks for your comments.
Andrea
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