Proteinase K treatment on viral RNA - (Dec/05/2007 )
Hi. I'm having trouble amplifying a gene using RT-PCR. I can amplify some part of the gene using internal primer so I'm quite sure that my template is ok. But amplifying the whole gene is always a failure. I've checked the primer using template from a different strain and it worked. My supervisor thinks maybe some protein bind to the RNA and make it impossible to amplify, and she suggested I treat my RNA sample with proteinase K. Does anyone has any idea how I'd do that? Thanks.
cDNA is the template for PCR so I assume the protein would be bound to the RNA and prevent cDNA sythesis not PCR. (Just semantics, don't mean to nitpick just clarify) What method are you using for RNA isolation and cDNA synthesis, ie: what primer is used for cDNA synthesis? What is the location of the internal primers with respect to the outside primers? Any other major differences -- huge size difference, GC rich region of the sequence etc.? I don't know about a protein remaining bound during isolation, I suppose it is theoretically possible but I have doubts about it, unless you have heard of such a thing before? If you decide to proteinase k treat be sure to add RNAse inhibitor as I assume that this enzyme, like others, cannot be purified completely clean of RNAses.
I extracted the viral RNA from allantoic fluid using a Promega kit. For 1st strand synthesis, i use specific primers. The outside primers (GC=48%) were designed to amplify the whole gene starting from ATG (~1.6kb), the internal primer (GC=56&) amplified cleavage site of the gene (~500bp). The Tm are similar. I haven't heard of protein contamination after phenol/chloroform either and my sample are clear, but we tried one-step RT, 2-step, with and without kit and it still doesn't work. Any idea?
I extracted the viral RNA from allantoic fluid using a Promega kit. For 1st strand synthesis, i use specific primers. The outside primers (GC=48%) were designed to amplify the whole gene starting from ATG (~1.6kb), the internal primer (GC=56&) amplified cleavage site of the gene (~500bp). The Tm are similar. I haven't heard of protein contamination after phenol/chloroform either and my sample are clear, but we tried one-step RT, 2-step, with and without kit and it still doesn't work. Any idea?
And you also have a positive control that works right? Did you RT the positive control at the same time as the experimental sample? I guess the first thing is to suspect is that you are not making cDNA that encompasses all primers, ie: the cDNA is dropping off so that the internal primers are synthesized but the 5' external primer is missing. There are alot of reasons for this that don't involve a protein remaining bound so I think it is easiest to test these first. The biggest reason is secondary structure at the 5' end blocks the extension by the RT this can be fixed by increasing the temperature that you RT at (many enzymes have a wide temp range but you may want to even purchase one that is specially made for high temperature RT) start with the RT you have and use the highest temperature that the manufacturer recommends... I would test this first, if you post your RT protocol/kit name etc. I can look to see if any other tricks come to mind...
Hope this helps!