Protocol Online logo
Top : Forum Archives: : Molecular Biology

RT-PCR: Band in no reverse transcriptase lane - (May/07/2007 )

Pages: Previous 1 2 

QUOTE (Horlem @ May 14 2007, 02:43 PM)
Not at all! Afaik, pure RNA should have a value of 2.1 (pure DNA of 2.0), RNA extraction with Trizol usually has a value of 2.0-2.1 by optimal starting material. I always include a DNase step, but just test a DNA sample, if you see 'your' band again, it would be the easiest way to get rid of it.

The tricky thing about Trizol is that phenol has the best absorbance at 270 - so you increase your spec reading by phenol contamination ... (I had good and reliable RT results in tricky tissues with spec readings below 1.8)
But for the problem of Cassio, protein contamination seems not so likely to me. I would guess on DNA contamination and that should be curable by a DNAse step.

-kr├╝melmonster-

I tried DNAse treatment, and it seemed to help. However, there's now a huge primer dimer band in addition to the RT PCR band. But at least there's no longer a band in the no-reverse transcriptase lane. The manufacturer's tech support suggests cleaning up the RNA after DNAse treatment and before going on to reverse transcription, even if the product literature suggests that it's not necessary.

But now I have another question. My experiments will involve 20+ different RNA samples, and the idea of DNAsing and cleaning up all those samples is daunting (but I'll do it if I have to).

So, here's my question: I'm using TRIzol to extract DNA. I searched this problem in the archives, in one thread somebody recommended performing a second (or even third) TRIzol / chloroform extraction to reduce/eliminate DNA contamination. Anybody know how well this works?

-Cassio-

Hi there,
I'm surprised that you think its less work to do a new trizol extraction than a Dnase step.

I find Dnase treatment is quite quick ~ around an hour.
I just use the Sigma product and it works well, doesn't degrade my RNA etc.

-smurray-

QUOTE (Cassio @ May 16 2007, 01:32 PM)
I tried DNAse treatment, and it seemed to help. However, there's now a huge primer dimer band in addition to the RT PCR band. But at least there's no longer a band in the no-reverse transcriptase lane. The manufacturer's tech support suggests cleaning up the RNA after DNAse treatment and before going on to reverse transcription, even if the product literature suggests that it's not necessary.

But now I have another question. My experiments will involve 20+ different RNA samples, and the idea of DNAsing and cleaning up all those samples is daunting (but I'll do it if I have to).

So, here's my question: I'm using TRIzol to extract DNA. I searched this problem in the archives, in one thread somebody recommended performing a second (or even third) TRIzol / chloroform extraction to reduce/eliminate DNA contamination. Anybody know how well this works?


You do not need a clean-up step after DNase treatment if you use Shrimp nuclease instead of DNaseI. Shrimp nuclease removes all double stranded DNA and is heat inactivated, which means you can throw it in with the primers and all,
and let it work at the same time as the RT, and heat inactivate by the same step that inactivates the RT.

-Gerd-

QUOTE (Gerd @ May 17 2007, 04:32 PM)
You do not need a clean-up step after DNase treatment if you use Shrimp nuclease instead of DNaseI. Shrimp nuclease removes all double stranded DNA and is heat inactivated, which means you can throw it in with the primers and all,
and let it work at the same time as the RT, and heat inactivate by the same step that inactivates the RT.


Well, Dnase I can be heat inactivated too right? What are the other advantages to use Shrimp nuclease? I think Dnase I will be cheaper right? laugh.gif

-timjim-

QUOTE (timjim @ May 17 2007, 02:48 AM)
QUOTE (Gerd @ May 17 2007, 04:32 PM)
You do not need a clean-up step after DNase treatment if you use Shrimp nuclease instead of DNaseI. Shrimp nuclease removes all double stranded DNA and is heat inactivated, which means you can throw it in with the primers and all,
and let it work at the same time as the RT, and heat inactivate by the same step that inactivates the RT.


Well, Dnase I can be heat inactivated too right? What are the other advantages to use Shrimp nuclease? I think Dnase I will be cheaper right? laugh.gif


You are not the one with the problem, no?
If you can choose between a cheap enzyme that destoys your results, and a new enzyme that may save both your time and your samples, you must have plenty of both if you are not willing to try something new.
But of cause, not my time nor my samples.

Shrimp DNAse is heat inactivated quicker and more complete than DNase I. Heating step can be deleteriouts for the RNA, so you do not want to heat inactivate at 75C if you can get away with lower temperatures, and get better end results.
Ambion did not come up with an alternative to heat inactivation of DNaseI, just for fun. Their method do however add another step.

DNase I also removed single stranded DNA, so you can not use it together with the primers.
This means you have to add an extra step in the procedure; no matter how you choose to get rid of it.
The major benefit of Shrimp DNase is reduced work.

I do not know about the prices at your vendor, but I would recommend asking for a free sample if you have never used it before. The enzyme is quite new on the market, have about the same properties as Kamchatka Crab DNase, but is much more affordable.

-Gerd-

haha.. nope, I am not the one with the problem. But just curious about both different enzymes. Sorry for being nosy wink.gif Just want to learn more things.

Thanks for the explaination though. wink.gif

-timjim-

Thanks for everyone's input!

The method to my madness is that I'm dealing with a largish number of samples, so I'll need a large amount of enzyme to treat them all, which my PI is loathe to pay for. Also, I really think it would be more time/money efficient to add/repeat a step or two during the extraction than to do a separate enzyme treatment. Maybe I'm wrong, but that's my thinking.

In troubleshooting this process I seem to be running into two camps. One group says "Gee, that's funny. I NEVER have DNA contamination in my RNA extracts." and the other group routinely treats with DNAse and wonders why I even question the need for it.

I'm painfully aware I don't have the best hands in the business, so I tend to concentrate on improving technique when I run into trouble like this. But if all else fails, I'll be sure to give some of the enzymes you've all suggested a try.

-Cassio-

Cassio,

you can try to perform more rounds of phenol:chloroform extraction (in the protocol section, the Miller protocol provides the concrete numbers). You could also try to use a column based system for RNA clean-up (e.g. RNeasy) but that's probably more expensive than the enzyme. As you told us about primer dimers occuring after the DNase treatment, maybe you try to decrease your primer concentration? If you have excess primers, unspecific amplification is much more likely to occur ... and there will allways be some primer left to amplify even the smallest rest of DNA!

-kr├╝melmonster-

Pages: Previous 1 2