Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

RT-PCR problem


  • Please log in to reply
4 replies to this topic

#1 dreamerbac

dreamerbac

    member

  • Active Members
  • Pip
  • 10 posts
1
Neutral

Posted 19 November 2009 - 09:49 AM

Hi,

I'm running RT-PCR for bacteria sample. My house keeping gene is 16s rDNA and I use stationary phase cultures. I have a no reverse transcritase control along with my cDNA product. So the problem is that I would have a wide difference (about 15 cycles) between the no RT control and cDNA when using 16s primers but almost no difference when i used the primers for genes of my interest. I understand that my RNA sample is probably not that clean since I do get amplification of the no RT control sample. But what would be the reason for what I see in the gene of my interest? Thanks!

#2 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 19 November 2009 - 09:54 PM

Hi,

I'm running RT-PCR for bacteria sample. My house keeping gene is 16s rDNA and I use stationary phase cultures. I have a no reverse transcritase control along with my cDNA product. So the problem is that I would have a wide difference (about 15 cycles) between the no RT control and cDNA when using 16s primers but almost no difference when i used the primers for genes of my interest. I understand that my RNA sample is probably not that clean since I do get amplification of the no RT control sample. But what would be the reason for what I see in the gene of my interest? Thanks!


Hi dreamer,
First, your RNA have (little) genomic DNA contamination. Try re-treat your RNA with DNAse.

If i not wrong, 16s is a repetitive gene. when there is no RT control in your PCR, your PCR amplification is using template from your very little genomic DNA contamination rather your cDNA. when you perform RT, the RNA from 16s (which i assume is plenty) was turned into cDNA, thus the PCR amplification is your cDNA + your little genomic DNA, which it turn out to be more amplification than the no-RT.

the no difference between no-RT and RT is because most probably your gene of interest is not being expressed. Thus the amplification is directly from your contaminated genomic DNA.

Hope this clears up your doubt.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 ciyiben

ciyiben

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 20 November 2009 - 01:12 AM

Hi dreamer

If you want to skip your DNA contamination, you can design your primers with one on two exons.
Hope i helped. Good luck <_<

#4 dreamerbac

dreamerbac

    member

  • Active Members
  • Pip
  • 10 posts
1
Neutral

Posted 26 November 2009 - 09:05 AM

Dear Adrian and Ciyiben,
Thanks a lot for your suggestions. I re-treated my RNA with DNase and this time the no RT control behaved. So it was due to DNA contamination. The gene of interest had good expression as well. Also it seems bacteria usually don't have exons in transcripts, as far as I know ;)
Dreamerbac

#5 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 26 November 2009 - 03:45 PM

Hi Dreamerbac,

Glad to hear that your RT-PCR is working. Keep up the good work and hope to see your publication some day in the future.

Adrian.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.