Best way to isolate viral mRNA for RT-PCR? - (Feb/25/2013 )
I am isolating Sendai virus mRNAs, from LLC-MK2 cells, and then quantifying using RT-PCR. I have used Roche's mRNA isolation kit (using magnetic beads); however I do not find the kit to be useful, as I run out the beads fairly quickly (and i'm not very trustful of reusing previously used beads). I was wondering if there are any suggestions for: Best mRNA isolation kits? (getting a lot of use out of it)
I have had excellent results with Ribozol. Cheap and efficient.
Thanks for the info! Will this work for specifically isolating mRNAs and not total RNA ?
No total RNA. Why do you need to isolate only mRNA? If you are doing RT-PCR, it will not matter what RNA is isolated. It will only rely on your GOI from your RT cDNA. You must know the viral sequence of your gene to perform RT-PCR. Are the viral primers you are using to similar to your LLC cells? Maybe I am missing something. If you absolutely need only mRNA, try the ambion mRNA pure kit from invitrogen.
Yeah, you're making a lot of sense! I all ready have the specific viral primers, used for the RT-PCR, but I am not sure if they are too close to the host cells. I actually never questioned why I can't just use total RNA, I'll bring it up to my P.I. tomorrow for sure.
Thanks for the help!