help - RT-PCR

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S.A.D.
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help - RT-PCR

Hello all,
I had isloated total RNA from frozen tissues using the Qiagen RNeasy kit.
My goal was to use this to make cDNA and then perform regular PCR in order to determine which isoforms of my protein were being expressed. For this I was using the Invitrogen -  'Superscript III One Step RT-PCR with Platinum Taq polymerase'.
I had designed gene specific primers but when I carried out the RT-PCR, I ended up getting multiple bands besides the band of the predicted size (which was also the brightest).
So then I decided to check for genomic DNA contamination by carrying out the same RT-PCR, except instead of adding the superscript III enzyme (for reverse transcription), I added only Taq DNA polymerase - ideally I should not see any PCR product, but in this case I did see 2 -3 bands - which I presume means that my primers were primming off of the genomic DNA.
I dont have much of RNA sample left, about 8microliters (of conc ~ 50ng/ microliter ) and with whatever I have left I just want to do a quick and dirty analysis to know if this is the right direction to pursue.
MY QUESTION - Can  I just add DNase I to my RNA sample, incubate it for a while at room temp, maybe inactivate the DNase I at 65C AND THEN DIRECTLY USE THAT SAME SAMPLE TO PROCEED ONTO RT-PCR using the Invitrogen kit???????????
Thanks
 

R Bishop
R Bishop's picture
S.A.D.,

S.A.D.,
In short yes you can. I've done it many times, just make sure you heat inactivate the DNaseI following the manufacturers instructions exactly. I always used Ambion DNase I that comes with a silica based gel to absob the DNase. Which you simply add and spin down after the incubation.
Question is this qPCR for your multi spliced gene?  We sort of lost track with one another on that topic.  Apologies.
 
Rus

S.A.D.
S.A.D.'s picture
Hey Rus,

Hey Rus,
Good to hear back from you. Yes, this is that very same project, you dont have to apologize to me for the communication gap cause if it hadnt been for your help, I wouldnt have even gotten started with this work in the 1st place.
So I just wanted to confirm, this Ambion DNase I that your talking about, is it the same as the TURBO DNA-free kit
This kit comes with the TURBO DNase which is a recombinant, engineered form of DNase I, and an 'Inactivation Reagent' which I presume is the silica based gel that you mentioned about.
So have you used this exact kit before to remove genomic DNA contaminants from the total RNA sample??????
I'm just trying to be sure cause I dont have much of the total RNA sample left for RT-PCR.
Thanks,
S.A.D.
 
 
 
 
R Bishop wrote:

S.A.D.,
In short yes you can. I've done it many times, just make sure you heat inactivate the DNaseI following the manufacturers instructions exactly. I always used Ambion DNase I that comes with a silica based gel to absob the DNase. Which you simply add and spin down after the incubation.
Question is this qPCR for your multi spliced gene?  We sort of lost track with one another on that topic.  Apologies.
 
Rus
Ivan Delgado
Ivan Delgado's picture
Another option you may want

Another option you may want to consider: if this RNA sample is very precious (i.e. you cannot really get more), and cost is not that much of an issue (in the order of $800), you could always take a fraction of your RNA (4 ul), DNAse treat it, perform the RT reaction, and then pre-amplify your cDNA using a kit like Applied Biosystem's Taqman PreAmp Master Mix kit. This way, you could start with a fraction of your RNA and still get plenty of cDNA for downstream applications.
 

S.A.D.
S.A.D.'s picture
Ivan,

Ivan,
Thanks for your post. Unfortunately I cannot afford to spend that much, $800 would really exceed the budget.
I'm waiting to hear back from Rus about the Ambion DNase, so in case you have any experience using it that would definitely be very helpful.
Thanks again
S.A.D.
 
Ivan wrote:

Another option you may want to consider: if this RNA sample is very precious (i.e. you cannot really get more), and cost is not that much of an issue (in the order of $800), you could always take a fraction of your RNA (4 ul), DNAse treat it, perform the RT reaction, and then pre-amplify your cDNA using a kit like Applied Biosystem's Taqman PreAmp Master Mix kit. This way, you could start with a fraction of your RNA and still get plenty of cDNA for downstream applications.
 
Jen_Floyd
Jen_Floyd's picture
 I generally use the Qiagen

 I generally use the Qiagen RT kit for my RT-PCR.  It includes a DNAse step in the beginning.  I imagine it doesn't matter the source of your DNAse as long as you heat inactivate before proceding with your RT reaction.

Ivan Delgado
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I agree with Jen that any

I agree with Jen that any DNAse should work fine for you. My experience is with Qiagen's RNAse-free DNAse (Cat. #79254). It works like a charm.

R Bishop
R Bishop's picture
Yep thats the one. Though the

Yep thats the one. Though the others suggestions shouls work fine as well.
 
Good luck SAD

Jason King
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Our entire lab has now

Our entire lab has now switched to using the M&N Nucleospin RNA II kits for total RNA preparation. This kit is relatively cheap and contains the DNAse, which after 15min incubation on the column, is washed away, so no need to heat inactivate.
 
You could also use intron-spanning PCR primers