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RT-PCR result !!


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4 replies to this topic

#1 noyara

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Posted 05 January 2011 - 12:21 PM

Hi all,

We extracted RNA from plant but the quality as gel electrophoresis shown is very bad , but after RT-PCR the band appear very clear and very nice,what that means?
how could we get very good result in RT-PCR where the extracted RNA is very bad?!! :blink:

And my second question is, why we used RNA and convert it to cDNA instead of using DNA directly?!! ;)

Thank you
Noor

#2 bob1

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Posted 05 January 2011 - 12:43 PM

You only need (theoretically) one copy of a gene to amplify it by PCR - so you can get a nice band from RT-PCR even when the RNA is low quality.

You use the transcribed RNA (RNA converted to cDNA) so as to have the gene without introns, which would make the genomic sequence huge for cloning and PCR (assuming you are trying to amplify the whole gene). If you are doing qPCR, then you are trying to estimate how much of the RNA is present at any given time, so you use the cDNA to estimate this.

#3 noyara

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Posted 05 January 2011 - 03:51 PM

You only need (theoretically) one copy of a gene to amplify it by PCR - so you can get a nice band from RT-PCR even when the RNA is low quality.

You use the transcribed RNA (RNA converted to cDNA) so as to have the gene without introns, which would make the genomic sequence huge for cloning and PCR (assuming you are trying to amplify the whole gene). If you are doing qPCR, then you are trying to estimate how much of the RNA is present at any given time, so you use the cDNA to estimate this.


Thank you very much for these answers, one more thing:
some postgrad student suggest that may this result is false positive come from binding the primer with DNA instead of RNA (extracted RNA contaminate with DNA), IS it possible? :unsure:

Also i have another problem ;) ,sometimes the bands (DNA marker and PCR product) not appear or appear but very light , how can i solve this problem? (i used, 1% agaros, 60 voltage,and add 1-3ul loading dye and rinse the gel with EtBr for 10 minutes)

Thank you
Noor :)

#4 ElHo

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Posted 06 January 2011 - 01:37 AM

First question: depends on the location of your primers. Primers for qPCR are usually designed not to amplify genomic DNA by spanning exon-exon-junctions.If your primers are located in the same exon, they will amplify both DNA and RNA with a pcr product of identical size. If your primers are located in different exons, you will get a longer pcr product for DNA compared to RNA, which can be checked by gel electrophoresis.

Second question: As both your standard and your samples are affected, it could either be the staining (not long enough, old staining solution) or the amount of DNA loaded (too little DNA, sample diffusion out of wells). Do you use the volume recommended for your standard?

#5 noyara

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Posted 06 January 2011 - 07:02 AM

First question: depends on the location of your primers. Primers for qPCR are usually designed not to amplify genomic DNA by spanning exon-exon-junctions.If your primers are located in the same exon, they will amplify both DNA and RNA with a pcr product of identical size. If your primers are located in different exons, you will get a longer pcr product for DNA compared to RNA, which can be checked by gel electrophoresis.

Second question: As both your standard and your samples are affected, it could either be the staining (not long enough, old staining solution) or the amount of DNA loaded (too little DNA, sample diffusion out of wells). Do you use the volume recommended for your standard?


Hi,
"which can be checked by gel electrophoresis." the expected size for this gene (pepcase) is 700bp, so in the gel also appear as 700bp when compared with marker, that mean the size is normal and not to long, is that what you mean..

and the 2nd thing is:
" Do you use the volume recommended for your standard?" Yes, exactly, and today in the lab something strange happen, i repeat every thing with different sample, and also the same problem happened (the band not appeared at all, just black gel!!) but i left the gel inside UV-doc for about 5-10 min then when i read it again the photo appeared very clear !! i said OMG, i get results and it was positive!! :lol:
i really don't know why...!!




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