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Best way to isolate viral mRNA for RT-PCR?

Isolating mRNA mRNA RT-PCR Sendai virus viral RNA

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4 replies to this topic

#1 ACN

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Posted 25 February 2013 - 05:24 PM

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)

Thanks!

#2 jerryshelly1

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Posted 26 February 2013 - 08:48 AM

I have had excellent results with Ribozol. Cheap and efficient.
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#3 ACN

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Posted 26 February 2013 - 07:55 PM

Thanks for the info! Will this work for specifically isolating mRNAs and not total RNA ?

#4 jerryshelly1

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Posted 26 February 2013 - 08:42 PM

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.

#5 ACN

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Posted 26 February 2013 - 08:50 PM

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!





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