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RT-PCR: DNA contamination, one vs two step


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11 replies to this topic

#1 noyara

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Posted 22 December 2010 - 12:13 AM

Dear All,

I have extracted RNA from plant leaves to convert it to cDNA, but I found there is contamination with DNA in RNA sample, Now can I use my extracted RNA to convert it to cDNA and then use primer for amplification the specific gene with this contamination or can't?
if yes, which is better the single step PCR or two steps?

Thank you
Noor

#2 BioMiha

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Posted 22 December 2010 - 04:37 AM

There is always a small amount of gDNA that is carried over in the purification steps. Most of the time people use intron spanning primers that bind only to the processed mRNA. Me, I prefer two step RT-PCR, first doing the RT and then using a dedicated kit for PCR.

#3 noyara

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Posted 22 December 2010 - 04:38 AM

17 views but no answer!! Why? :blink:

Please I really need your help, anyone have information in this field? :(

any help will be appreciated

#4 phage434

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Posted 22 December 2010 - 05:34 AM

Ambion and others make either DNAse for removal of genomic DNA contamination, or columns with bound DNAse for removal of DNA. Depending on the intron location and numbers, you may be able to amplify a gene product without purification, relying on the smaller length of the spliced transcript.

#5 noyara

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Posted 22 December 2010 - 06:19 AM

There is always a small amount of gDNA that is carried over in the purification steps. Most of the time people use intron spanning primers that bind only to the processed mRNA. Me, I prefer two step RT-PCR, first doing the RT and then using a dedicated kit for PCR.


Thank you very much for your helping, may you recommended me for suitable kites to order?

And also I want to add this: when i check the DNA in my RNA sample in nano-spectrophotometer I found the DNA concentration higher than the RNA concentration
as follow:
RNA concentration:0.34 ug/ul
DNA concentration:97.0 ug/ul
Is that consider OK for pcr?

Many thanks

#6 David C H

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Posted 22 December 2010 - 06:51 AM

Qiagen and Zymo Research sell DNase/buffer combinations and both sell RNA clean up kits.

Your sample appears to be mostly DNA and that is unusual for an RNA isolation procotol -- have you checked the quality by running it on a gel?

If the RNA concentration is 0.34 ug/ul (which is 340ng/ul) you definitely have enough RNA. I recommend checking your sample on a Bioanalyzer or a standard agarose gel (run at high voltage for 15-20min); I am concerned that because the RNA concentration is much lower than DNA that your RNA may be degraded.

#7 BioMiha

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Posted 22 December 2010 - 07:24 AM

NanoDrop measures just the absorbance of your sample and calculates the concentrations of your RNA or DNA based on the ratio of A260/A280. However, both DNA and RNA absorb at these wavelengths, so I would be cautious interpreting only the ug/ml values. Also in my experience many things that you carry over in your RNA prep will absorb in the 260/280 nm range, so I never trust the read-out. As David C H said, you are better checking your sample on a gel. RNA travels completely differently in a gel than gDNA, however you must be cautious, because single stranded nucleic acids bind EtdBr differently than double stranded (much less), so you cannot compare these two directly. For RNA preps we use the RNeasy kit from Qiagen and in my hands it's the best i've tried. It also has a gDNA elimination step, but the manufacturer says that you cannot eliminate gDNA completely, especially if you have a lot of tissue.

#8 noyara

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Posted 22 December 2010 - 07:55 AM

Ambion and others make either DNAse for removal of genomic DNA contamination, or columns with bound DNAse for removal of DNA. Depending on the intron location and numbers, you may be able to amplify a gene product without purification, relying on the smaller length of the spliced transcript.



In the protocol that i used for RNA extraction (RNA kit from Geneaid) mentioned about DNase but my supervisor inform me this is optional step, because of that i wander whether it's possible to amplify specific gene with presence of DNA ,
Many thanks for your answering

#9 noyara

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Posted 22 December 2010 - 08:03 AM

Qiagen and Zymo Research sell DNase/buffer combinations and both sell RNA clean up kits.

Your sample appears to be mostly DNA and that is unusual for an RNA isolation procotol -- have you checked the quality by running it on a gel?

If the RNA concentration is 0.34 ug/ul (which is 340ng/ul) you definitely have enough RNA. I recommend checking your sample on a Bioanalyzer or a standard agarose gel (run at high voltage for 15-20min); I am concerned that because the RNA concentration is much lower than DNA that your RNA may be degraded.


I prepared gel as described in sambrook book chapter 7 , MOPS, and using DNA ladder, I found nice band from RNA sample, but the DNA ladder not appear, I don't know why, may be MOPS not suitable for DNA,
any how I prepared normal procedure for agaros gel 0.8% with TBE,10V/cm, then I found the RNA sample bands start from about more than 1000bp and reached to 500bp, when I compared with DNA ladder (from fermentas 100bp DNA ladder)

I think it's not RNA, I'm I right?

#10 noyara

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Posted 22 December 2010 - 08:16 AM

NanoDrop measures just the absorbance of your sample and calculates the concentrations of your RNA or DNA based on the ratio of A260/A280. However, both DNA and RNA absorb at these wavelengths, so I would be cautious interpreting only the ug/ml values. Also in my experience many things that you carry over in your RNA prep will absorb in the 260/280 nm range, so I never trust the read-out. As David C H said, you are better checking your sample on a gel. RNA travels completely differently in a gel than gDNA, however you must be cautious, because single stranded nucleic acids bind EtdBr differently than double stranded (much less), so you cannot compare these two directly. For RNA preps we use the RNeasy kit from Qiagen and in my hands it's the best i've tried. It also has a gDNA elimination step, but the manufacturer says that you cannot eliminate gDNA completely, especially if you have a lot of tissue.


The Nano-spectro. that used is from IMPLEN,
You can check this website:
http://www.implen.com/

Kindly can you explain to me this sentence : "RNA travels completely differently in a gel than gDNA" how can i differentiate between both?

And thank you very much

#11 David C H

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Posted 22 December 2010 - 08:17 AM

check this link for images of RNA run on an agarose gel:

http://www.ambion.co...pp/rna_gel.html

If you can see the ribosomal bands and your RNA isn't smeared to the bottom, you definitely have good enough RNA for RT-PCR. Partially degraded RNA can be fine if you are amplifying 100-200bp fragments.

Since you are new to RT-PCR, I recommend starting with two-step as it is more reliable. If you know your primers work with one-step, you can go ahead with that.

#12 noyara

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Posted 22 December 2010 - 08:37 AM

check this link for images of RNA run on an agarose gel:

http://www.ambion.co...pp/rna_gel.html

If you can see the ribosomal bands and your RNA isn't smeared to the bottom, you definitely have good enough RNA for RT-PCR. Partially degraded RNA can be fine if you are amplifying 100-200bp fragments.

Since you are new to RT-PCR, I recommend starting with two-step as it is more reliable. If you know your primers work with one-step, you can go ahead with that.


I'll take into consideration these notes,
Many thanks




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