How to differentiate between RNA and DNA

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

HELISA
Death Adder
Death Adder
Posts: 67
Joined: Sat Feb 25, 2006 12:55 am
Location: Illinois

How to differentiate between RNA and DNA

Post by HELISA » Thu Sep 13, 2007 1:12 am

Hello Everybody!
I have a problem to solve, and I have few ideas how to approach it, but maybe you Guys have better solutions?
I have two samples, one of them contains RNA, while the other DNA. I don't know which one is which. What are the possible ways to differentiate between them?

My ideas:
1) absorbance measurement A260/A280 for DNA 1.8, for RNA 2.0, pretty lame
2) RNaze A treatment which would digest RNA leaving my DNA sample intact, simple agarose gel would show me which is which
3) DNase digestion leaves RNA intact cleaves DNA, similar approach to the previous one :!:
4) restriction endonuclease, cuts just dsDNA, leaving RNA intact...

Any other ideas?
Image
Image

User avatar
mith
Inland Taipan
Inland Taipan
Posts: 5345
Joined: Thu Jan 20, 2005 8:14 pm
Location: Nashville, TN
Contact:

Post by mith » Thu Sep 13, 2007 2:04 am

how long are they?
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr

User avatar
MrMistery
Inland Taipan
Inland Taipan
Posts: 6832
Joined: Thu Mar 03, 2005 10:18 pm
Location: Romania(small and unimportant country)
Contact:

Post by MrMistery » Thu Sep 13, 2007 5:37 am

i may be wrong, but from what i know, EtBr only interacts with DNA, not RNA. If this is indeed true then put EtBr and stick in into the electrophoresis machine.
Also, PCR would deffinetly work in amplifying only the DNA, not the RNA
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

User avatar
mith
Inland Taipan
Inland Taipan
Posts: 5345
Joined: Thu Jan 20, 2005 8:14 pm
Location: Nashville, TN
Contact:

Post by mith » Thu Sep 13, 2007 6:39 am

No, etbr works on RNA too. In PCR's we'd see a solid signal and then a fuzzier one further down stream.
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr

User avatar
MrMistery
Inland Taipan
Inland Taipan
Posts: 6832
Joined: Thu Mar 03, 2005 10:18 pm
Location: Romania(small and unimportant country)
Contact:

Post by MrMistery » Thu Sep 13, 2007 8:57 am

ok, like i say i wasn't sure. but pcr would work
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

SororSaudade
Death Adder
Death Adder
Posts: 65
Joined: Thu Nov 16, 2006 3:54 pm
Location: Portugal

Post by SororSaudade » Thu Sep 13, 2007 9:43 am

If you can spare some of you sample, I think the RNase or DNase treatment could be a very good and simple way of knowing that.
I'm not an expert though..

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Thu Sep 13, 2007 1:26 pm

SororSaudade wrote:If you can spare some of you sample, I think the RNase or DNase treatment could be a very good and simple way of knowing that.
I'm not an expert though..


However lame solution 1 is not bad either if you cannot afford to lose the samples. of course this suppose a good purity of the samples. So I second SororSaudade if you can afford to do it (and I suspect you can, since this sounds more like lab/homework question than a real life experiment).
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

HELISA
Death Adder
Death Adder
Posts: 67
Joined: Sat Feb 25, 2006 12:55 am
Location: Illinois

EtBr

Post by HELISA » Thu Sep 13, 2007 2:18 pm

I'm sure EtBr intercalate with secondary structures of RNA, so that wouldn't work for my purpose...
How about the use of restriction enzymes? Do you guys know the enzyme which would cut almost every kind of DNA leavind RNA intact?
Image
Image

blcr11
Viper
Viper
Posts: 672
Joined: Fri Mar 30, 2007 4:23 am

Post by blcr11 » Thu Sep 13, 2007 5:13 pm

DNase I will work and so will RNase A, but make sure you use quality enzymes; no DNase contamination in the RNase A and vice versa. RNA is more sensitive to alkaline hydrolysis compared to DNA (which is why it is alkaline lysis you use to purify plasmid DNA). RNA usually runs way out front of typical plasmid DNA or restriction fragments on agarose gels and shows up as a fuzzy spot at really low MW—you can even run it off the gel if you run too long—after staining with EtBr. If there’s a lot of RNA contamination—like if you forget to add RNase to the lysis buffer when preparing your plasmid DNA—the RNA staining will be very intense and raise the background staining in the entire well. I think there are some RNA-specific dyes around—Northerns were once stained with acridine orange(?) or something like that if I remember correctly. You can do the 260/280 ratio thing, but it is often hard to interpret. The rule of thumb that “ratios of 2 or more imply RNA” is often correct, but not always.

User avatar
jesusfreak0318
Garter
Garter
Posts: 3
Joined: Fri Aug 10, 2007 5:53 pm
Location: Arkansas
Contact:

thymine

Post by jesusfreak0318 » Sat Sep 15, 2007 2:16 am

one of them (DNA I believe) has thymine while the other (i think it's RNA) has something else
God's more than the laughter or the stars in the heavens.

User avatar
mith
Inland Taipan
Inland Taipan
Posts: 5345
Joined: Thu Jan 20, 2005 8:14 pm
Location: Nashville, TN
Contact:

Post by mith » Sat Sep 15, 2007 2:23 am

lol...........
Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr

HELISA
Death Adder
Death Adder
Posts: 67
Joined: Sat Feb 25, 2006 12:55 am
Location: Illinois

LOL

Post by HELISA » Mon Sep 17, 2007 10:54 pm

:lol:
that's a good observation!
Image
Image

Post Reply

Who is online

Users browsing this forum: No registered users and 8 guests