RNA extraction and DNase treatment - (Jan/27/2015 )

Dear all,

I'm doing molecular biology work. I do RNA extraction and my RNA is contaminated with gDNA. I used phenol:chloroform method to carry out RNA extraction. After the DNase treatment i found out that my RNA was also degraded. I found out this through nanodrop reading and also the band intensity in the gel electrophoresis. The DNase treatment method i used was 1.Mix : RNA - 5ug, Buffer 10x- 5ul, DNase- 5ul, Add dH2O up to 50ul 2. Incubate in room temperature for 10 min 3. Add 1ul of EDTA (25mM) 4. Incubate 65 deg C for 10 min 5. Chill at ice for 1 min Then run gel. 

My question is :

1) Can you help me with DNase treatment?

2)Is it necessary to carry out DNase treatment since we only want to amplify the gene of interest through PCR?

3) Can we proceed to do RT-PCR and PCR without doing the DNase treatment?

4) What is the drawback if we continue RT-PCR and PCR without DNase treatment? How does it affect the final result? 

Please help

Two possible problems:

1) presence of RNAse in your preparation. Make sure you are using RNAse free  reagents, clean tips and tubes, wear gloves, etc. Also, I'd suggest adding 1 ul of SuperRNasin to your prepared RNA.

2) Insufficient removal of metal ions prior to 65C heating. I would add  more EDTA to make sure that all of the Mg++ ions were removed prior to a heat step. The heat step may not be necessary. Normally I run DNAse reactions for 30 minutes at 37, then chill,'

Depending on your primer designs, gDNA removal may not be important. If your primers amplify across an intron, for example, the primers will amplify only spliced mRNA. You should definitely run an RT- control to verify that you are not amplifying gDNA regions.

How do you see gDNA contamination? How do you run RNA on a gel? As far as I know you are not supposed to run RNA on typical DNA agarose gels.
In most cases primer design should overcome gDNA contamination anyway (intron spanning primers). Intronless pseudogenes in the gDNA would be a problem, presumably.

You did not specify the manufacturer of your DNAse (this is essential!), but the protocol appears similar to ours and we are using Invitrogen, DNAse I. However, you use 5x less EDTA. Our manufacturer suggests to use >2mM EDTA (final). 

We do not take extreme precautions with our RNA (e.g. DEPC treated water, RNAse inhibitors), yet qPCRs work ok.

@phage: If you do not heat-inactivate DNAse, won't it cleave your cDNA?