15 replies to this topic

[The Question]

Dear all

I'm really unhappy with my DNA isolation at this moment since I cannot get ride of RNA from the sample, which I've heard will interfere with DNA during PCR. For removal of RNA I did RNAse treatment and then phenol extraction and CIA (chloroform+isoamyl alcohol) extraction. The DNA was precipitated using NaOAC and absolute ethanol, pelleted, washed with 70% ice-cold ethanol, dissolved in TE solution and the concentration was measured using Nanodrop. The ration 260:280 is all the time around 2. I repeated RNase treatment in same sample but no improve.
To look for the presence of other stuffs along with DNA, I also did ran agarose gel with the sample. Here is also the photo of the gel (sample and ladder). It seems from gel my DNA is not pure. I require some help, please!

[DocFlow]

The Question, on Nov 20 2009, 12:38 PM, said:

Dear all

I'm really unhappy with my DNA isolation at this moment since I cannot get ride of RNA from the sample, which I've heard will interfere with DNA during PCR. For removal of RNA I did RNAse treatment and then phenol extraction and CIA (chloroform+isoamyl alcohol) extraction. The DNA was precipitated using NaOAC and absolute ethanol, pelleted, washed with 70% ice-cold ethanol, dissolved in TE solution and the concentration was measured using Nanodrop. The ration 260:280 is all the time around 2. I repeated RNase treatment in same sample but no improve.
To look for the presence of other stuffs along with DNA, I also did ran agarose gel with the sample. Here is also the photo of the gel (sample and ladder). It seems from gel my DNA is not pure. I require some help, please!
KR002176.JPG

Have you tried to do PCR on your DNA/RNA extract and checked whether possible RNA interferes with your reaction? Usually it's the other way around, that's why many automated DNA extraction protocols don't have an extra RNase step. I would have tried the PCR just to give it a check.

The ususal problem with nanodrop measurements on DNA samples containing RNA is that is measures both, which again may give you a false high "DNA" concentration and a too rough dilution of the DNA afterwards. Have you tried measuring DNA by picogreen or other fluorescence methods?

[The Question]

DocFlow, on Nov 20 2009, 01:00 PM, said:

The Question, on Nov 20 2009, 12:38 PM, said:

Dear all

I'm really unhappy with my DNA isolation at this moment since I cannot get ride of RNA from the sample, which I've heard will interfere with DNA during PCR. For removal of RNA I did RNAse treatment and then phenol extraction and CIA (chloroform+isoamyl alcohol) extraction. The DNA was precipitated using NaOAC and absolute ethanol, pelleted, washed with 70% ice-cold ethanol, dissolved in TE solution and the concentration was measured using Nanodrop. The ration 260:280 is all the time around 2. I repeated RNase treatment in same sample but no improve.
To look for the presence of other stuffs along with DNA, I also did ran agarose gel with the sample. Here is also the photo of the gel (sample and ladder). It seems from gel my DNA is not pure. I require some help, please!
KR002176.JPG

Have you tried to do PCR on your DNA/RNA extract and checked whether possible RNA interferes with your reaction? Usually it's the other way around, that's why many automated DNA extraction protocols don't have an extra RNase step. I would have tried the PCR just to give it a check.

The ususal problem with nanodrop measurements on DNA samples containing RNA is that is measures both, which again may give you a false high "DNA" concentration and a too rough dilution of the DNA afterwards. Have you tried measuring DNA by picogreen or other fluorescence methods?

Thanks for reply

I used isolated DNA for qPCR. I get the required product. But the problem is I don't get efficient result. My supervisor was saying for the preparation of standard curve with isolated DNA first instead of plasmid DNA. The low efficiency of my qPCR result could be due to the presence of RNA in my sample, I think.

[DocFlow]

What if you physically isolate/cut out the correct band from your gel and do another PCR on it? If you still see the additional band and smear on the next agarose gel the problem might be insufficient PCR reactions and not interfering RNA.

[The Question]

DocFlow, on Nov 23 2009, 09:36 AM, said:

What if you physically isolate/cut out the correct band from your gel and do another PCR on it? If you still see the additional band and smear on the next agarose gel the problem might be insufficient PCR reactions and not interfering RNA.

Hi DocFlow

Thanks again for reply. That could be something I still can try. By the way the gel I've shown above is just from isolated DNA but not from PCR product.

[gogreen]

hi TQ, Can you tell us what was the method you used for DNA isolation? What was your starting material, cells/tissue??

[Prep!]

can u elaborate what do u mean when u say low effficiency of the PCR?? are u saying sensitivity is complromised? or hight Ct values?
Are u doing a Q-PCR?.. as doc said... rna in your sample will not effect the amplification reaction most of the times.

[The Question]

Pradeep Iyer, on Nov 23 2009, 11:54 AM, said:

can u elaborate what do u mean when u say low effficiency of the PCR?? are u saying sensitivity is complromised? or hight Ct values?
Are u doing a Q-PCR?.. as doc said... rna in your sample will not effect the amplification reaction most of the times.

I used phenol extraction method for C elegans. I meant indeed there could be some problem in sensitivity due to the presence of RNA in the sample. I heard from my supervisor as well low sensitivity could be caused due to the presence of high amount of RNA as well. In my qPCR for instance, sometimes I get higher Ct values for lower dilutions.

[Prep!]

well ya tat means your RNA might be interfering with the reactions.. or alternatively due to the presense of RNA you are quantifying the DNA wrongly (over estimating it) and hence adding less amount than what shud be added so the lower sensitivity!!
So at hgher dilutions that is more concentration the Ct values are unaffected??
How does your positive control behave???

Edited by Pradeep Iyer, 23 November 2009 - 03:11 AM.

[The Question]

Pradeep Iyer, on Nov 23 2009, 12:10 PM, said:

well ya tat means your RNA might be interfering with the reactions.. or alternatively due to the presense of RNA you are quantifying the DNA wrongly (over estimating it) and hence adding less amount than what shud be added so the lower sensitivity!!
So at hgher dilutions that is more concentration the Ct values are unaffected??
How does your positive control behave???

I don't know why I get slightly higher or similar Ct values for higher dilutions (compared to lower dilution). And at this point I was thinking RNA as a probable problem. What do you mean by positive control here? I was willing to use isolated DNA to make my standard curve instead of plasmid DNA. So, I used isolated DNA and negative control for my qPCR.

[Prep!]

ya you can very well use the isolated DNA to prepare the standard curve. that will lessen any errors due to the standard curve of a DNA without contamination if the contamination is your problem.
It is some times possible that at higher concentrations of template, it masks the background but at lesser conc it cannot due to low amount of template!!

[The Question]

Pradeep Iyer, on Nov 23 2009, 12:28 PM, said:

ya you can very well use the isolated DNA to prepare the standard curve. that will lessen any errors due to the standard curve of a DNA without contamination if the contamination is your problem.
It is some times possible that at higher concentrations of template, it masks the background but at lesser conc it cannot due to low amount of template!!

Thank you Pradeep for your replies.

I'll give some try to look at what really is wrong and probably will come to forum later.

[Prep!]

Ya sure do that
BEst luck!!!

[warsel]

This may be rather naive but: what stops you from adding some more RNAse A to your sample?
I don't think that should interfere much with your PCR reaction..

[The Question]

warsel, on Nov 24 2009, 06:00 PM, said:

This may be rather naive but: what stops you from adding some more RNAse A to your sample?
I don't think that should interfere much with your PCR reaction..

Hi Warsel

I tried with higher volume of RNA as well though I've understood the small quantity of RNase is also sufficient for the degradation of RNA. But the problem remains the same. Now I think the RNA-nucleotides are also precipitated during centrifugation and that gives smear on my gel. Can that be the problem? Any suggestions are appreciated.

Thanks