4 replies to this topic

[dreamerbac]

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!

[Adrian K]

dreamerbac, on Nov 20 2009, 01:49 AM, said:

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.

[ciyiben]

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

[dreamerbac]

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

[Adrian K]

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.