I am looking to profile miRNAs (via qpcr) within clinical samples (tissue & blood) and have been fascinated towards using the polyadenylation based strategy using SYBR green primarily because of lower cost than taqman assays. Since I am the first in my lab to use this, I am trying to garner a public opinion from users who have opted for this technique in the past/present:
A. What is your impression of this technique (polyadenylation of mirs followed by qpcr using SYBR) - in terms of amplification efficiency, sensitivity, specificity, ease, etc - over the taqman based assays?
B. I have observed several kits from numerous companies that provide the polyadenylation polymerase, RT polymerase and SYBR? Which ones have you used and would recommend?
I analysed miRNA from murine liver tissue samples using Trifast for RNA extraction, polyadeylation, RT-PCR using poly-A adapter and SYBR based QRT-PCR.
The real time PCR performance regarding amplification efficiency and specifity was comparable to the measurement of common cDNA. But a thorough design of miR-primer is necessary, to be aware of unspecific binding of similiar structured miRNAs. In my case, it was necessary to optimize the template amount, template concentration was in the end much lower than used in common real time assay. Unfortunately, I have no experience with Taqman based assays.
In my case 2.5 U of poly(A) polymerase from New England Biolabs (NEB) was sufficient for modification of 1 µg RNA. After polyadenylation RNA was purified with RNA clean&concentrator kit (Zymo Research). Reverse Transcription PCR was performed with ABI High-capacity RT-PCR kit with 0.5 µg poly(T) adapter instead of random primer. Small nuclear RNA U6 (RnU6) was used as internal control and quantitative PCR was performed with SensiFast lowROX kit from Bioline.
@Jan
Many thanks for your detailed response. All the same has been very helpful in gaining a better insight. I have the following questions though:
A. By any chance, did you also work with blood/urine/stool/FFPE RNA for the above mentioned process? I just wanted to understand if low/bad quality RNA input affects the quality of qpcr.
B. I was also curious to understand the sensitivity of this technique in detecting small RNAs present at low concentrations within in your test samples.
Looking forward to hearing more from you in this regard.
I'm sorry, I've just analysed and extracted frozen tissue samples. Since I was not aware of some low quality extractions or sampes, I can tell you my experiences. For low quality samples always was the chance to analyse them. The expression (Ct) could be lower, but in line with the appearence of internal standard. That was common for normal cDNA and miRNA products of reverse transcription. Really bad quality RNAs, for instance if you see just breakdown products on agarose gel, has to be excluded generally.
The sensetivity of these measurements was also a surprise for me. I analysed in total 12 different miRNA. Some were well known to be expressed in liver tissue, but not all of them. Most Ct values were in the range of 20-32. Just in the case of 1 miRNA I obtained just weak signals. And, there was still the possibility to increase the template amount. Of cause. my experiences are restricted to a limited number of tissues and microRNAs.
Thanks a ton Jan!
All the information provided by you has been very helpful & encouraging for my work!
Thanks again!
Hi, I have been using these kits for a few years now for the analysis of serum and tissue. The ploy A method has some great advantages when you have low RNA (e.g. Serum) as you don't reverse transcribe each mirna of interest as you do for single Taqman assays. We have also compared our results with Taqman assays and been able to show we get the same result. I now use the miScript system from qiagen.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA