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qPCR and miRNA isolation - (Apr/27/2014 )

Hi everyone,

 

In my project, I will first try to silence a gene and then will look for its effects on miRNAs in cells(eukaryotic). 

Basically, i will use lipofectamine reagent and specific siRNA to silence the gene(say X gene).

 

1) To show that silencing is accomplished, I will isolate total RNA, convert to cDNA and run a qPCR reaction. Then,

 

2) I will analyse miRNAs in the X gene-silenced cells. For that, I need to isolate miRNAs and send them to RNA sequencing.

 

So my question is, how can I do 1 and 2 using the same sample? 

 

I need to do them using the same sample, because i need to look for specific miRNAs in the X gene-silenced cells. Same sample or another possible way, I am waiting to hear your suggestions. 

 

Thanks in advance.

 

Regards,

 

Fatih

-ofbaltaci-

Your first step in both cases is RNA isolation. You can split the RNA sample after isolation. You need to do an RNA isolation which preserves the small miRNA fragments. This probably is an acidic phenol or Trizol isolation.

-phage434-

Thanks. I thought about this solution, but I just couldn't be sure whether the amount of RNA will be enough for both processes.

 

How about splitting cells after silencing process? It doesn't sound scientific, though. But maybe we can use one half of the cells for the confirmation of silencing and the other for miRNA analysis. My concern is that can we make sure that the cells we are gonna use for miRNA isolation are X gene-silenced?

-ofbaltaci-

High throughput sequencing needs very small amounts of RNA/DNA, so I doubt if this is a problem. I would find splitting the sample after RNA isolation more convincing as a reviewer.

-phage434-

High throughput sequencing needs very small amounts of RNA/DNA, so I doubt if this is a problem. I would find splitting the sample after RNA isolation more convincing as a reviewer.

Thanks a lot.

-ofbaltaci-