Hello! I am doing a microRNA over expression experiment. One of it is targets is ATM, which is down regulated upon correct transfection/ over expression of the microRNA of interest.
Can I detect ATM by RT-PCR or only by western-blot? I have been doing through western-blot but I would like to know if it's reliable to test it by RT-PCR too.
Hi there,
If you would like to check the ATM expression by qPCR, you can go ahead and check the expression. Also, for the same experiment you can perform RT-PCR to check the DNA of RT PCR. But, here, you will be required to standardise the PCR cycle to . Start with minimum of 15 and load only 8ul/50ul reaction of DNA. You can put another 5 cycles of the same reaction.
And for the same experiment you can validate the protein level using western data. I have perform some experiments like this. Message me if you need any further help. Good luck.
Dear Bruna, sometimes mRNA level and protein level are not concordant and even do not correlate. As Pratibha said, you can realize both: Western and PCR. You may count it like a different experiments and compare the results.
If you want to conduct gene expression estimation with a real time PCR be carefull with reference genes. If you have a questions, please do not hesitate to ask. If You have enourh time you can visit https://www.gene-quantification.de/ to receive perfect results.
Since miRNA affect expression on post-transcriptional level (in the cytoplasm not the nucleus) most often protein level is more affected than mRNA level. However, in some cases also mRNA expression is modulated (maybe more indirectly via regulatory loops in cellular expression control).
I would propose to test effects of your miRNA on its targets on protein level (e.g. via WB or ELISA).
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