Hi, I am doing RNA isolation from Thremoanaerobacter kivui, it a thermophilic bacteria. I am using Qiagen Rneasy Mini kit. I also tried classical method using Trizol reagent. I have always obtained DNA contamination even after Dnase treatment. I am using Turbo Dnase. Does anyone suggest me how to get rid of DNA?
Hello Surbhi
How do you know that you have DNA contamination? Electrophoresis or PCR? How sever is the contamination? Using this DNase will take up to 30 min to degrade your contamination. If you already followed manufacturers instruction, you may need to use more DNase. Have you tested your DNase functionality? You may need to make sure it is working. To test that you need some extracted DNA, and run a gel after DNase treatment to see validate the functionality of your DNase.
Good luck
Greetings,
DNA contamination is something every single person who works with RNA has to deal with. There are some things you can do to work this out.
1. Try to do a double RNA extraction with TRIzol, using the first extraction as template for the second one. Even then, it's probable that you might have gDNA around.
2. Increase the DNase-RNA ratio, adding more DNase per ug of extracted RNA.
3. If the latter doesn' t work, check your DNase specifications for optimum temperature range. Set your thermocycler to the upper temperature, for the same amount of time.
That should solve it. Good luck
PD. When working with TRIzol, try leaving around 100-200 uL of the upper aqueous phase behind. It's better to lose some RNA ng than getting biased results from RT-qPCR.
Hi Surbhi,
First of all you should check your RNA samples for presence DNA contamination. In my case, I use qPCR - I added 1 uL of RNA samples instead of cDNA. If I see the signal, I have the information that my RNA samples were contaminated with genomic DNA (the low CP values give information about high amount of DNA contamination).
Additionally, I had the same problem with DNA contamination. I based on Blood/Cell RNA Mini Kit from Syngen. To avoid DNA contamination you have to use additional DNase I treatment (in Kit from Syngen this is an additional purification stage). When I used double DNase I treatment, my RNA samples were pure and all worked properly.
Good luck!
Dear colleagues (Surbhi Jain, Patrycja Ogonowska, Adán Valenzuela Castillo, Ali Javadmanesh) I agree with your suggestions, but I would like to add one more way.
Of course, Trizol and related kits is the best way to receive quite clear RNA. BUt in the majority of cases it is not possible to eliminate DNA fully. DNAse treatment can help to decrease Ct but frequently only decrease. Moreover DNAse treatment leads to partial degradation of RNA even in column-baseb protocols and in liquid phase of course (after Trizol).
That's why You can try oligos that can't amplify DNA. It is possible. Not easy ) but it is possible. In this case DNA contamination is not a problem after Trizol. You can save time and increase RNA quality.
Exon/exon placment of primer is a good idea, but be carefull in some cases you may touch with abnormal sequences (pseudogenes) that contain part or full sequence of mRNA.
Ali Javadmanesh Thank you for the response and suggestion. I did PCR to check DNA contamination. Yes, the Dnase is functional, I have verified that. I have also increase the time of Dnase digestion, but unfortunately, that is also not working for me.
Adán Valenzuela Castillo Thank you for the response and the suggestions.
Regarding your suggestions:
1. Today I am trying double RNA extraction with TRIzol. I hope it will work.
2. I tried increasing the Dnase volume. But, it did not work.
3. Yes, checking the optimum temperature is a good idea. I am incubating at 37 degrees. Any suggestion, how much temperature I should increase ?
Yes, I am leaving some amount of upper aqueous layer behind.
Patrycja Ogonowska Thank you for the suggestion and response. I tried doing double Dnase treatment. But its not working for me.
Andrei S. Babenka Thank you for the suggestion and response. Sorry, I do not understand, how to design primers that can't amplify DNA.
It is quite popular action. I provide the principle in attached picture. If you have a questions please do not hesitate to ask me.
They are also known as intron spanning primers @Surbhi Jahin, It's Also a Good suggestion. To give you a better answer, can you tell me what kind of DNase are you using?
Dear Surbhi Jain
I think this link below is useful to solve your problem
https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination
Adán Valenzuela Castillo I tried double RNA extraction with Trizol. It did not work as well. I am using Turbo Dnase from thermofischer.
Try to perform double TRIzol extraction, followed by diluting your samples to a concentration of 10 μg/ 50 μL, as suggested by thermofisher, or less, add double or triple Turbo DNase volume (4 to 6 U), and incubate at 37 °C by 30 min. I believe such a hard treatment should be enough to digest any trace of gDNA present on your samples. Good luck.