Hello Sreedevi M J

How have you designed the primers for the MicroRNA? You can try setting up a real time experiment and run the products on an agraose gel if the primers are working fine. Also, try to check the process for cDNA preparation as the cDNA preparation for MicroRNA requires a different approach.

Hope this helps. Good luck.

Sakshi Agrawal Ma'am I designed my primers using miRprimer software, also used Qiagen's miRCURY LNA RT Kit for the cDNA conversion.

Dear Sreedevi M J,

The problem with the microRNA primers is due to the short nucleotide length they are more prone to Nucleases.

The following steps may be useful to you to encounter the issue:-

1. Confirm your primer sequencing does they are flanking and binding to pre-target.

2. Make sure your extraction of miRNA does not contain inhibitors and that all reagents are within the expiry date.

3. Confirm by taking OD the check the purity, quality, and concentration of RNA. Due less stable nature of RNA may lead to the breakage of single standards.

4. Conversion of cDNA always prefers to go with less volume. And again It depends on your application.

5. Ensure the sample is fresh to obtain the desired miRNA, Check for extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD).

6. Immediate conversion is always recommended to avoid chaos.

7. Check the gel percentage and you did not mention whether your control and Ladder have migrated well.

In summary, Ensure the primer sequence target site of your sample. As Sakshi Agrawal mentioned you should take care with your isolation part and also try to use RNA Rescue solution to use it as a surfactant. Check the yield of RNA and Conversion of cDNA and the temperature that you're maintaining for the process. The preparation of gel for the electrophoresis is a promising step to encounter such problems. I hope this information may be helpful. My best wishes in your future endeavors.

Srinivas Chaganti Sir, 2% gel was used for the electrophoresis. I checked my cDNA conversion using the reference gene, It showed bands in gel, also my ladders were correctly migrated. Moreover, I have checked a range of Tm from 52 to 66, but I didn't see any bands.

If you post your gel image that will be more helpful to all to give the right solution to your problem. Thank you.

Srinivas Chaganti these are my gel images sir.

If you are working with miRNA then 2% gel is not enough due to the shorter length of miRNA and its PCR products, try 10-15% PAGE.