I am currently using the "qSTAR miRNA qPCR Detection System" protocol. I have isolated my miRNA cDNA, with my control concentration being 0.852 μg/μl and my transfected concentration being 0.637 μg/μl. The first step of the qPCR protocol says to transfer 2 μl of the miRNA cDNA to the qPCR plate. However I do not know if my concentrations are too high for the qPCR because the protocol does not set a maximum or recommended concentration of the miRNA cDNA. Please let me know any information that may help me continue my qPCR!
I am not sure the concentration you mentioned is cDNA or miRNA. If cDNA then concentration is too high for conducting qPCR. It is giving good amplification and melting curve using cDNA conc. 50 - 70 ng/μl i. e. 0.05 - 0.07 μg/μl . Try to do qPCR by diluting your cDNA.
- miRNA is a tricky one when we have tried to amplify from patient blood
- In general you cannot actually measure the concentration of any cDNA by spec because of unincorporated dNTPs; Only by fluorimetry. Always better to measure the starting concentration of RNA and make sure they are the same
- Better still with miRNA because of the tiny amounts you are dealing with, It is better to pre amplify before performing RT
- See my attached protocol
The thumb rule of almost every PCR kit is that you can change the volume of water to add more cDNA and viceversa, just stay with the recomended final reaction volume. Any way, you can do qPCR with 10 ng of DNA, but in the case of cDNA you always get a mixture of cNTP's, several species of RNA and cDNA. So you have to go a little generous with the amount of cDNA, you can try going from 50 to 200 ng to find the optimum Rx conditions, running various concentratios in the same plate. Don't forget that too much template genetic material can inhibit de DNA polymerase reaction.
Ujjal Kumar Nath, Laurence Stuart Dawkins-Hall, Adán Valenzuela Castillo Thank you all so much!
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