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Contamination in -rt


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#1 ni_labgirl

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Posted 26 February 2009 - 02:20 AM

Hi all,

I have been having a number of problems with my transient transfections (MCF-7 cells). I have been getting bands in my rt-pcrs for the -rt samples of the transfected cells (I never have this problems with any of my other samples).
My supervisor says this isnt surprising as we are adding a load of DNA to the cells but hasnt really offered any other advice, I have tried a few different kits and seperate DNase treatment but cant seem to shift it!

So I just wondered if anyone had similar problems and if you managed to eliminate it? I can only think now that I will have to reduce the amount of DNA but didnt want to impact on my transfection efficiency.

Thanks,

:)

#2 Trof

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Posted 26 February 2009 - 05:47 AM

You can redesign your primers so they span an intron (one half of primer in exon1 the other in exon2) and DNA wouldn't amplify at all.

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#3 ni_labgirl

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Posted 03 March 2009 - 04:17 AM

You can redesign your primers so they span an intron (one half of primer in exon1 the other in exon2) and DNA wouldn't amplify at all.


Thanks Trof, I have went back and looked over my primers (learned alot more about why we design them they way we do in the process!) and my second (reverse) primer does span an intron.

So I wondered why I was still getting amplification - so now I have checked and the gene I am transfecting in is an origene trueclone 'human full-length cDNA clone' which we hade a glycerol stock of - could this be the problem in that i am actually transfecting in cDNA rather than DNA and my primers cant distinguish - if this is the case is there any way around this??

Sorry if I am askin simple questions but all help is greatly appreciated.

#4 mikew

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Posted 03 March 2009 - 02:50 PM

Hi,

You need to be a little more specific.
You are transfecting the cDNA for gene X into a cell, purifying RNA
and amplifying what? Are you amplifying gene X as well? Or gene Y?

If you transfect a cDNA encoding gene X into cells and purify the RNA
using a column you will get a boat load of that plasmid coming along with your
RNA and his will amplify in your PCR reactions.

It is difficult to get rid of. The only thing I have seen done is to isolate your RNA and THEN do a full DNAse digestion of your RNA. Then phenol/chloroform (ph5.4) and precipitate. Or rerun through a column
And then do the RT reaction.

Alternatively do a luciferase assay or a knockdown.

#5 ni_labgirl

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Posted 05 March 2009 - 02:49 AM

Hi,

You need to be a little more specific.
You are transfecting the cDNA for gene X into a cell, purifying RNA
and amplifying what? Are you amplifying gene X as well? Or gene Y?

If you transfect a cDNA encoding gene X into cells and purify the RNA
using a column you will get a boat load of that plasmid coming along with your
RNA and his will amplify in your PCR reactions.

It is difficult to get rid of. The only thing I have seen done is to isolate your RNA and THEN do a full DNAse digestion of your RNA. Then phenol/chloroform (ph5.4) and precipitate. Or rerun through a column
And then do the RT reaction.

Alternatively do a luciferase assay or a knockdown.


Thanks Mike,

I am transfecting in a gene 'X' from the cDNA plasmid prep followed by RNA extraction using Qiagen's RNeasy kit (with a step where I add DNase to the column and leave at room temp for 15mins) following this a standard RT step.

To confirm the transfection was successful I was doing a semi-quantitative RT-PCR to check that gene X was increased in my transfected cells before I would move on to check other genes to see if there expression was up/down in the transfected cells - however I havent progressed any further as I keep getting these bands in my -rt for the cells transfected with gene x.

But then again, is it really important if I do a western to show that the gene x is definitely expressed higher in the transfected cells and its not just carry over from the pasmid?




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