I am facing some problems while performing rt-PCR of mir205, 196b, 375, 21 and u6snrna. Every time the CT value of u6snrna is more than 33 and sometimes it is closer to 37. I have extracted whole RNA and I have done small RNA enrichment from FFPE samples. In both cases the CT value of u6snrna is not improving. On the other hand, the CT values of the target miRNAs are always within 20 to 30 cycles. I am using miRVANA as extraction kit and taqman probe for rtPCR. My RTpcr protocol is 50 for 2min, 95 for 10 min, 95 for 30 sec, 62 for 3min followed by 40 cycle. 60 for 1min. Auto delta enabled. My samples consist adeno and squamous cell carcinoma.
From vast experience with RT-qPCR of miRNAs and reference genes (and I mean hundreds of thousands of reactions and many projects over dozens of miRNAs on several platforms), I can say there is something fundamentally wrong with your U6 assay, since its abundance is huge and the Ct values are usually at least 10 cycles below abundant miRNAs. It will not be improved by increasing RNA in RT (which is non-linear anyway, and you risk saturating the reaction. Always stay within manufacturer-recommended range.), or increasing cDNA in the RT (you are about 15 cycles off normal Ct, so there is no way to compensate this much). Moreover, both above solutions will similarly decrease the miRNA Ct. Something is seriously WRONG, not just SUB-OPTIMAL. I would not do small RNA enrichment from FFPE as a lot of it is degraded so you are biasing your samples according to the degree of overall degradation. RT-qPCR should work with total RNA. Moreover, if your enrichment is too harsh, you may be losing U6 in the "large" RNA fraction. Not hugely probable, but not impossible.
Easiest possible explanation is human error, which we have made in the past: check which species assay you ordered! While mouse miR-205 assay will perfectly well amplify mouse miR-205, the snRNA assays do not cross between species. If all assays are correct, check your U6 assay on an unrelated sample, preferably total RNA from a cell line. The assay could have degraded or got contaminated with RNase or some inhibitor. Unlikely, but not unheard of. Is the U6 problem consistent across batches of probes? If so, I would get a different snRNA reference, like U44. If the problem persists, check your RT-PCR conditions (they don't quite make sense the way they are listed - the 30" denaturation should be withing the cycling not before it, but that could be the website uploading something in a fragmented way). Try separating the RT from the qPCR. I prefer one large RT reaction with multiplexed primers for all my genes of interest, then split it across multiple gene-specific qPCRs. This way I know for sure the RT itself has worked. Also allows you to incorporate an RT-negative control. The U6, for example, should amplify DNA as well as RNA.
Lots of other options to troubleshoot. Keep us posted. But most importantly, don't settle for a patch solution as your data analysis may be completely wrong as a result.
Best of luck!
Anna
You must increase the amount of RNA in your RT. Increase the volume of RT in the PCR have only a weak effect.
@Stephane Roche...... you mean I should use more amount of cDNA while final reaction volume will be the same.
In the qPCR volume, you must increase 10X to obtain increase of 2-3 cycles. You must work in the level of your RT to increase the amount of your wanted template.
I have done a lot of qPCR for miR205 specifically in prostate cancer. We had good results using the Qiagen miRNeasy FFPE kit, using RNU6B as the housekeeper. As a positive control, normal tissue from the same patient was used, miR205 is strongly downregulated in prostate cancer (this is not the case for all epithelial tumour types).
With FFPE material, the yield allways depends on the fixation conditions, whether people used buffered formalin and how much time there was between the operation and the fixation, how long the tissue had been soaking in the formalin,... Once fixed, it's pretty stable though.
From vast experience with RT-qPCR of miRNAs and reference genes (and I mean hundreds of thousands of reactions and many projects over dozens of miRNAs on several platforms), I can say there is something fundamentally wrong with your U6 assay, since its abundance is huge and the Ct values are usually at least 10 cycles below abundant miRNAs. It will not be improved by increasing RNA in RT (which is non-linear anyway, and you risk saturating the reaction. Always stay within manufacturer-recommended range.), or increasing cDNA in the RT (you are about 15 cycles off normal Ct, so there is no way to compensate this much). Moreover, both above solutions will similarly decrease the miRNA Ct. Something is seriously WRONG, not just SUB-OPTIMAL. I would not do small RNA enrichment from FFPE as a lot of it is degraded so you are biasing your samples according to the degree of overall degradation. RT-qPCR should work with total RNA. Moreover, if your enrichment is too harsh, you may be losing U6 in the "large" RNA fraction. Not hugely probable, but not impossible.
Easiest possible explanation is human error, which we have made in the past: check which species assay you ordered! While mouse miR-205 assay will perfectly well amplify mouse miR-205, the snRNA assays do not cross between species. If all assays are correct, check your U6 assay on an unrelated sample, preferably total RNA from a cell line. The assay could have degraded or got contaminated with RNase or some inhibitor. Unlikely, but not unheard of. Is the U6 problem consistent across batches of probes? If so, I would get a different snRNA reference, like U44. If the problem persists, check your RT-PCR conditions (they don't quite make sense the way they are listed - the 30" denaturation should be withing the cycling not before it, but that could be the website uploading something in a fragmented way). Try separating the RT from the qPCR. I prefer one large RT reaction with multiplexed primers for all my genes of interest, then split it across multiple gene-specific qPCRs. This way I know for sure the RT itself has worked. Also allows you to incorporate an RT-negative control. The U6, for example, should amplify DNA as well as RNA.
Lots of other options to troubleshoot. Keep us posted. But most importantly, don't settle for a patch solution as your data analysis may be completely wrong as a result.
Best of luck!
Anna
Dear all, I have tried all the suggestions provided by you guys, though there are no improvements in Ct value.
One thing more, I have extracted whole RNA from normal blood sample, and put the assay. but result is same. Ct value of U6 snRNA is still more than 33.
Dear Sangeet, in my experience U6 has a very low endogenous expression, I believe it is hard to get a better ct from it. You can try RNU-48, that perfectly works across the samples. Good luck.
Hello Sangeet
How much concentration (ng / ul) of Total RNA in your sample?.
what is ratio of 260/280 and 260/230?
How many conc ( ng ) / how many uL you taken ( Total RNA) to convert cDNA?
While converting cDNA did you add any RNA spike?
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