I use Trizol method to isolate RNA from frozen PBMCs. Sometimes, I get 260/280 ratio of ~1.8 at which I get slight amplification in my no-RT control, indicative of genomic DNA contamination. I therefore want to give DNAse treatment to the extracted RNA and then re-extract pure RNA. Literature that I have gone through suggests that I use phenol-chloroform extraction and ethanol precipitation to re-extract my RNA.
Can somebody please give me a detailed protocol for the same?
You are right. Follow the instructions that come with your DNase (ie how much to use for the amount of RNA you have). DNase is very easily denatured so be careful to mix gently. You will then need to get rid of the DNase from the mix so add phenol/chloroform 1:1(be sure to use acidic phenol for RNA, not the one you would use for DNA) adding equal amount to the volume of RNA mix you have. Mix well. Spin 13k rpm 5 minutes. Extract aqueous phase, avoiding interface. Precipitate DNA by adding 1/10 volume of 3M Sodium Acetate pH 5.2 and 2.5x volume absolute Ethanol. Leave at -20 for 2 hours. Spin 13K rpm 4oC 15 minutes. Remove liquid carefully. Wash pellet with 70% Ethanol and spin again. Air dry pellet.Now you have clean RNA. Resuspend in nuclease-free water and heat 60oC for 10 minutes.
Following on from what Andrei has added, if I'm isolating a large amount of RNA from one source then I use Trizol and DNase treat exactly as above but if I need only small amounts of RNA from lots of different treatments then the kits are much more convenient and they have a DNase step included. They are expensive though. I use the Qiagen one.
Dear Ashish,
Yes I agree with Andrei Nikonov, It is better to isolate total RNA with RNA isolation kits such as Trizol, RNeasy Mini Kit, RNAXPlus, ... , then treat the total RNA with DNase or other clean-up kits. It's good for you. let me know if you have further question.
good luck
Ashish, I think that the best way to purify your DNA is the acetate sodium precipitation, I agree with Amanda, this protocole is efficient and you will not affect your DNA quality and concentration.
I'm agree with suggestions from Amanda. Another additional could be perform a treatment with Turbo DNAse from Ambion after you have resuspended your RNA (I think that it is your case). It is very powerful DNAse that even digest DNA in high salt concentration and after you do not need to make Phenol-Chloroform because you can use beads which are able to bind to DNAse in order to get rid off it from your tube (included in Turbo Dnase Kit). Sometimes, we are using it for downstream applications such as RT-qPCR. Good Luck!
First heat inactivate the DNase by keeping at 65degrees for 10minutes. Then,
you can simply precipitate the RNA from the solution by adding 1/10th volume 3M Na-acetate and 2.5-3 volumes 100% ethanol and by keeping it either at RT for 10-15minutes or at -20degrees for 1hour. Then, spin down the RNA at maximum speed and give a 75% ethanol wash. You can go for phenol-chloroform extraction when you need very pure RNA. For normal RT-PCR or qRT-PCR experiments, this protocol works for me.
I have always used on column DNAse treatment for DNA free RNA for microarray analysis. Using this method I always get RIN scored around 9.8-10. It is really easyt an yields good quality RNA. I wouldn't bother messing around with Trizol. Try the Ambion or Qiagen (exact same kit) with the Qiagen RNAse free DNAse. You just cook the column with the bound RNA with DNAse for 15 min or so at room temp then carry on with the washes.
Hi
please find the link
http://fg.cns.utexas.edu/fg/protocol__DNAse_treatment_of_RNA.html
The DNase can only handle reactions with an RNA concentration at most 200 ng/µL, as calculated in the reaction specified above.
1.Remember to add components in this order: water, RNA, buffer, DNase
2.Mix gently and centrifuge briefly
3.Incubate at 37ºC for 20 - 30 min
Your RNA has now been treated with DNase, and you must extract the RNA from the mixture to avoid destroying any cDNA you generate in RT-PCR with DNase that might still be left in the sample.
RNA Extraction
1.You may either proceed in a one step extraction with 1:1 phenol/chloroform solution, or two steps with acid phenol and then chloroform (in all cases, add volumes equal to your RNA sample’s volume).
2.Transfer the aqueous phases to new tubes. Avoid the interface. If you are performing the two step protocol, add the chloroform here, and extract the aqueous layer.
3.Add 3M sodium acetate (pH 5.2) equal to 1/10th the volume of your sample to your RNA sample.
4.Add 2.5 volumes of 100% ethanol.
5.Incubate at -20ºC for 1.5 hours.
6.Spin at max speed in the 4ºC centrifuge for 10 minutes.
7.Decant the 100% ethanol, and wash the nucleic acid pellet with 70% ethanol twice.
8.Dry the pellet and resuspend in nanopure water.
9.Nanodrop the resulting RNA sample to check for contamination. Proceed with RT-PCR.
did it work? :) dealing with the same and information is scarce indeed to perform this.
@Ashish Kumar which method did you use in the end? There is lots of helpful advice here but I am unsure which to try.
Hello! I am currently experiencing a similar issue. I wanted to ask if you use Acid-Phenol:Chloroform:IAA (125:24:1 pH 4.3, I presume), and then a second extraction with Chloroform:IAA (24:1)? Or Do you just proceed with the aqueous layer directly from the Acid-Phenol:Chloroform:IAA step and add the NaOAc and ethanol precipitate?
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