Dear Scientists,
I have some Kidney and Liver tissue samples infected with Minus-Single Strand RNA Viruses (RNA - ). However, I need a determined Gene from Viral RNA Genome to amplify. I have some problems ahead...
1) Is it possible to amplify the Viral RNA target gene directly from tissue sample?
2) Do I need to concentrate the virus titre in the tissue sample?
3) How can I get sure about both tissue and viral capsid digestion in upstream levels?
Almost Protease doses mentioned in protocols are utilised for tissue digestion, not capsid digestion simultaneously. Should I add more Protease to the solution for capsid digestion? Or I should apply it in a different step?
Regards
Sayadi
Hi,
First, you have to extract viral RNA from tissue samples. There are different commercial kits for extraction viral RNA from tissues. In our laboratory we mostly use Qiagen products. RNeasy Mini Kit has a satisfactory results when we use it for extraction viral RNA from different tissues such as liver, kidney, spleen, lung, lymph node, ...After extraction you can use your RNA samples for RT-PCR assay.
Dear Sevik,
How can I extract it in a manual way? Without kit?
I guess i can extract total RNA, and then run a pcr for specific RNA gene from virus, is it possible?
Hi, probably you can extract it using phenol or TRIzol method and then run PCR with specific RNA primers.
good luck
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