Did u see ur extracted mirna in urea page gel???

Use Trizol followed by etoh pptn. Use pre designed primers from applied biosystems. Works every time for us. Do not use the qiagen kits for rna clean up, you'll loose your miRNA. Something happened, the turbulent solution you describe is odd.

I never used your protocol so I don't know what's going on here. We always used either the Ambion miRvana kit like in the previous answer (for fresh tissues or cells) or the Qiagen RecoverAll kit for FFPE samples. Then used the Applied system for both RT and q-PCR (TaqMan probe). Always worked for us. As for Trizol-based methods of RNA extraction, there was a discussion here a few days ago about a group in Korea who had to retract a paper after they found that, with low sample cellularity, Trizol was introducing very significant bias in the miRNA content.

Just use Trizol and NCode™ miRNA First-Strand cDNA Synthesis Kit from Invitrogen, design the primers according to the protocol. It works well!

Clean up your reaction after Poly-A- w/ the Phenol Chloroform extraction then do your CDNA synthesis.

Interesting. I had not seen that paper. I may look for it if I can. I would like to know what their cell numbers were.

why you don't try LNA primer and exiqon SYBR green kit ? you need a positive control to sure that primers work .

Thank you all. I am sure about realtime master mix. I also tried ABI master mix (SYBR Green). I used Qiazol for all sample, so I have to use Qiagen miRNasy kit. I have purchased cDNA synthesis kit and all of primers from applied biological materials (ABM), so is not possible to change matrials. I will clean up the reaction after poly A tailing. Thank you again.

Dear Dipta Sengupta,

Why I have to extract miRNAs from gel? I do not want to use them for transfection or microinjection.

Hi Mariano,

Here the link to the news page with that story. From there you can to the original paper and the new one where they describe how they became aware of the issue. Quite interesting as it shows how we can discuss for quite some time about results that are nothing more than technical artifacts.

https://retractionwatch.wordpress.com/2012/07/13/group-retracts-microrna-paper-after-realizing-reagent-was-skewing-results/?goback=.gmp_154833.gde_154833_member_134262698

Dear Vahid

I don't have any experience of working with your procedure for miRNA extraction and cDNA synthesis. In this way, I extracted total RNA by Trizol and I used an Iranian kit (PARSGENOME) to prepare all materials related to cDNA synthesis and specific primers for real time PCR. Until now, everything is OK.

But all in all, first you should be sure that all materials related to your purchased kit don't have any problem. Unfortunately, purchased kits are sometimes destroyed during transfer from abroad to our country. Then, I suggest that you extract total RNA by Trizol again and follow the procedure as you mentioned. Maybe it can solve your problem.

Good Luck

May be there is problem in poly A tailing as well as in cDNA synthesis. You should better use stem loop RT PCR protocol for the synthesis of cDNA, consequently the end point PCR. there are lots of paper in these regards. If you find further problem, feel free to contact with me.

Describe a new, highly sensitive and specific PCR approach for quantitation of microRNAs (miRNAs): the miRCURY™ LNA microRNA PCR system. The method, which allows detection of 10 copies of miRNA, is enabled by the use of Locked Nucleic Acids (LNA™). The LNA-conferred sensitivity facilitates accurate detection of miRNA expression in a single cell.

HI

I think the stem -loop primer technique in miRNA expression analysis with qPCR is the most accurate and cheapest method.

Hi all, I could get the acceptable results, I found that mncl2 in poly A tailing kit interfers the cDNA synthesis process, once I cleaned up the sample after poly A tailing and got the result. In another sample I did not used mncl2 for tailing process, both results were acceptable!! Thank you

Try NCode (http://products.invitrogen.com/ivgn/product/MIRC10). It works!

Hi,

I am having the same problem with the Q-PCR and cDNA synthesis! how did you clean up the poly A? By phenol Chloroform?

Thank you for your help.

Marty

I have the same question: how did you choose to clean up the reaction after poly A tailing. So far I have read: heat inactivation or addition of EDTA.

Since 2010… I use Trizol standard method to isolate total RNA. After, I strongly recommend NCode kit for cDNA synthesis.  Finally, I use specific miRNA-primers along with NCode universal primers to perform real time PCR, using SYBR GREEN. It always works! 

Hi Vahid, I used the exact same protocol as you mentioned. Total RNA extraction followed by phenol-chloroform, ethanol precipitation then proceeding to cDNA synthesis and real-time PCR. But the problem that I have is the amplification curve reaches threshold around 3-5 cycle which usually should between 20-30. Do you quantify you RNA after ethanol precipitation? If you do, how much RNA did you add to synthesize cDNA and after cDNA synthesis, how do u dilute your cDNA sample for real-time PCR? Thank you!