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Gene silencing and miRNA isolation

siRNA silencing miRNA isolation

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#1 ofbaltaci

ofbaltaci

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Posted 26 April 2014 - 09:46 AM

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



#2 medibio

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Posted 06 June 2014 - 09:09 AM

Hi,

 

You can isolate total RNA include miRNAs to use Trizol reagent, so you can also obtain miRNAs







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