Hello everyone please i'm a newbie in molecular biology. i have use a protocol of carrying out Dnase and Rnase treatment on a sample before the extraction using Purelink Viral RNA/DNA kit but still my RNA quality and concentration remains low and not fit for further RNA library preparation and sequencing whereas the DNA is fit for DNA library construction and sequencing.
Anyone has tips on what i can do to improve my resulst?
i maintain the near best laboratory standard for RNA/DNA extraction to avoid Rnase contamination.
please share your experience. Thank you.
Hello Alex Obeten
I would prefer doing DNase and RNase treatment after RNA and DNA extraction.
For DNA samples:
You may make the DNA volume to 200ul in TE buffer (pH 8.0). Add 10ul of RNase A (10 mg/mL) and incubate 37°C for 1 hour. To check for the complete removal of RNA you may check small aliquot (5ul) on an agarose gel with no treatment control. Run gel for 15-20 minutes. If there is no traceable RNA, proceed to the next step. If significant amounts of RNA are still present, add another 10ul of RNase A and repeat the incubation.
For RNA samples:
You may include DNase I treatment step after RNA extraction to remove contaminating DNA. However, the DNase must be removed or inactivated in the RNA sample. This may be achieved by Proteinase K digestion (150 µg of proteinase K per milliliter of sample) for 30 min at 50°C, followed by phenol/chloroform extraction and NH4OAc/EtOH precipitation. This extraction specifically cleaves the DNase I and precipitates the DNA, while the RNA remains in the aqueous phase and can be recovered by a subsequent precipitation.
However, if you are using heat (heating at 75°C for 5-10 minutes) to inactivate DNase I, you may add EDTA to a final concentration of 5mM before heating. If EDTA is not added, the RNA will undergo chemical scission when heated.
For confirmation, run an agarose gel, DNA contamination will be visible as a smear or band of fragments considerably larger than the RNA.
Best.
Malcolm Nobre Thank you so much for you kind response. my challenge is i'm trying to extract both DNA/RNA from the SAME sample of 200ul. This is my experimental protocol using purelink viral RNA/DNA kit
1. Add Dnase 1 and Rnase to the sample incubate at 35 degrees for 30 mins
2 increase to 75 degrees for another 30 mins and then follow the rest of the protocol from the manufacturers.
my challenge is the concentration of the RNA is often lower than the DNA and the sample still has contamination of Rnase after i analyse the 260/280 ratio and 260/230.
so my confusion is should i apply the Rnase and Dnase treatment at the end after the elution step with 50 ul Rnase free water?
What is the purpose of the DNase and RNase treatment, and what is the rationale for doing them on the same sample? I'm really struggling to follow the purpose of the experiment, which makes it hard to give advice.
Laura Leighton is right. Alex Obeten I am confused too. DNase and RNase treatment on the same sample!
Laura Leighton the treatment of EVs sample with Rnase or Dnase is to eliminate the extra vesicular RNA and DNA. This has no effect on the RNA and DNA contained within EVs and viral particles inthe sample.
Hi Alex, I'm responding to your message here because I think it's more beneficial to the community when answers are available in public rather than in private messages. I don't believe anything I've said below is likely to be confidential re your aims or samples, but if I'm wrong, please let me know and I will delete this post.
If you want people to be able to give meaningful advice, it really helps to have adequate information about what your inputs, outputs and aims are. My understanding of your current protocol is:
1) You are isolating both extracellular vesicles and also intact viral particles from feces. (How much feces? Could more be used?)
2) This isolate is in a volume of 200uL and is the input for the DNA/RNA extraction kits.
3) You are treating this isolate with both DNase and RNase to destroy extravesicular/extraviral nucleic acids. (Which DNase and which RNase?)
4) You are then incubating the sample at 75 degrees for 30 minutes. (What is the purpose of this step, destroying the DNase and RNase?)
5) After this, you do DNA and RNA extraction according to the manufacturer's protocol
6) At the end, you have 50uL of eluted RNA at a concentration of 7ng/uL, and your sequencing provider says this isn't enough and won't sequence it.
I can see some potential issues with this protocol based on my understanding, which I am not sure is correct. If I am wrong about any of the steps or their purpose, please tell me so we can get closer to a solution!
The main issue I see is that 75 degree 30 minute incubation. Do you know that your viral particles and extracellular vesicles are able to survive this incubation temperature for this length of time? If you are cooking them and they are breaking and releasing their contents, this will reduce your RNA yields. Also, 75 degrees will not destroy RNases. I think the better option would be to stop the DNase/RNase digestion by flooding the reaction with EDTA (final concentration 50mM), and then using a harsh lysis buffer (eg phenol/chloroform) to get rid of the DNase and RNase. I think it is very likely that the first step of your extraction protocol would be adding lysis buffer - is this accurate?
The next thing that can probably be changed is the elution step. Does your sequencing provider say whether the AMOUNT or CONCENTRATION of the RNA sample is the problem? If the issue is that the total AMOUNT is too low, you will need to look at ways to decrease sample loss (eg, eliminating an overly long and hot incubation) or increase sample input (eg, use more poop.) If the issue is that the CONCENTRATION is too low, you can decrease the elution volume so that the same amount of RNA is more concentrated, making sure that you do not exceed the minimum elution volume for the column.
Finally, as I said in my message, I think you could consider pushing back on the sequencing provider here or even switching providers. 7ng/uL of RNA in a 50uL elution is 315ng assuming 45uL recovery. This is heaps, there are many RNA sequencing kits on the market that can make quality libraries with far less RNA than this, and you need to ask your provider whether they can use a low input library prep kit, and if not, why not. It might cost extra, but it's very much within the realm of possibility.
@Laura Leighton thank you for your kind response and recommendations. I appreciate your apt commens as i find them most helpful. Thank you.
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