Hello guy, I need your help to solve my problem in experiment. I got sample as culture cell line. I have to detect whether virus infection or not in this cell culture sample (pharmaceutics product). I should test the presence or not of RNA virus from bovine/porcine in these cell lines by quantitative real-time PCR method.
My plan is: I will find the specific sequence for these RNA viruses to design primer, probe and plasmid (that I will use as positive control when I running RT-qPCR, and make standard curve). In this case, when I perform RT-qPCR I need RNA or DNA to running.
My question here is: In this case, how can I isolate RNA or DNA of virus from culture cells? Or anyone have experience to perform this kind of experiment before can tell me what should I do in this case?
I have never do this kind of experiment before so I am so confused. Thank you so much for your help.
The safest way is to use commercial kits such as "MagMAX Viral and Pathogen Nucleic Acid Isolation Kits (RUO)". For example, this kit has been used for the isolation of SARS-CoV-2 and other infectious disease pathogens.
The other well-known method is Phenol-chloroform extraction (see details below):
Materials and Reagents:
- Virally infected cell culture
- Cell culture media and reagents
- Phosphate-buffered saline (PBS)
- Cell scraper or trypsin
- Centrifuge
- Lysis buffer (proteinase K, SDS, EDTA)
- Phenol-chloroform-isoamyl alcohol
- Chloroform
- Ethanol
- DNA precipitation reagents (e.g., isopropanol, sodium acetate)
- 70% ethanol
- TE buffer (Tris-EDTA) or other suitable DNA storage buffer
Procedure:
Remember that the specific protocol may vary based on the virus you're working with, the cell type you're using, and the DNA extraction kit or reagents you prefer. Always follow biosafety guidelines and precautions when working with viral samples, and adapt the protocol to your specific needs and conditions.
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