I am performing snRNA Seq from mouse pancreas. Usually RNases is a major problem when working with pancreas. I was planning to use DEPC treated water to make solutions since DEPC treated water is better than normal water when working with RNA. But I was wondering if this will in anyway interfere with downstream applications? Any suggestions would be helpful. Thank you!
For this experiment, you need to think about where the RNases are coming from. DEPC treatment of water removes any RNases that are in the water, and the solution is then autoclaved to destroy the DEPC so it can't interfere with downstream applications. DEPC treated water is just water without RNases in it - using it gives you confidence that you are not adding RNases to your reaction with the water, and it should be a standard practice to use DEPC treated or ultrapurified water for all RNA experiments. But DEPC water is not an RNase inhibitor, it cannot eliminate RNases that get into your experiment some other way. Pancreas tissue contains a really high level of endogenous RNase, so the vast majority of the RNase in your experiment is actually coming from your samples themselves. This means you will need to be very careful during extraction of your RNA.
Tips for RNA extraction and usage from RNase-rich tissues:
- Ideally process the tissue fresh, within minutes of the animal's death. Drop into ice cold buffer immediately after removal from the animal, and process immediately.
- If this is not possible, chop the tissue into small pieces and immediately snap freeze in liquid nitrogen or dry ice. When ready to extract, drop frozen tissue pieces into ice-cold lysis buffer and begin homogenising immediately, there MUST be NO period of time for which the tissue is thawed but not homogenised.
- Process only a few samples at a time. The more you do at once, the more 'sitting around time' each of them has, and with pancreas it needs to be very minimal.
- Use 2-4x the standard amount of Trizol or lysis buffer per weight of tissue.
- If using Trizol or another phenol/chloroform extraction method, extract twice. (Follow the protocol up to the phase separation step, then mix the aqueous phase with more Trizol, and start again.)
- When you are done working with tissue, thoroughly clean your work area with RNase decontamination solution (eg RNase ZAP or RNaseAway or look up one of the homemade recipes online.) Do this while your samples are spinning, don't wait until after the extraction procedure is over.
- Keep everything as cold as possible, and consider adding ribonuclease inhibitors early (eg, as soon as you elute your RNA - RNase inhibitors into the tube.)
- Use ribonuclease inhibitors in all downstream reactions. RNA library prep kits usually include them in the buffers, but for pancreas I'd add an extra 0.5-1uL of RNase inhibitor to each reaction at the start, and then again after any high temperature enzyme inactivation steps.
Hope this helps!
DEPC-treated water reduces the risk of RNA degradation by ribonucleases. To inactivate DEPC, the solution is autoclaved at 37 ° C for at least 2 hours. So no, DEPC-treated water does not interfere with downstream applications.
Hello Shilpa Patil
Yes, DEPC treated water does interfere with downstream applications when working with RNA, if used at high concentration and not inactivated by autoclaving.
When used at a concentration of 0.1% DEPC solution and autoclaved for 15 minutes it can be assumed to be DEPC-free. Autoclaving inactivates DEPC by causing hydrolysis of diethyl pyrocarbonate. CO2 and ethanol are released as reaction by-products. A faint ethanol smell may linger after autoclaving. This is caused by the ethanol by-product combining with trace carboxylic acid contaminates and forming volatile esters.
However, using increasing DEPC concentrations can result in high levels of residual DEPC or DEPC by-products in solution that can inhibit some enzymatic reactions or chemically alter (carboxy methylate) RNA. It has been documented in studies that DEPC by-products in RNA samples can inhibit in vitro translation reactions as well.
Best.
In my experience, we can use unopened, sterile and RNase free water from any High class system or equipped with supplying RNase free water.
The use of DEPC have an important role in reducing the level of rnase contamination in the final stage of RNA extraction, but other sources of exposure to this enzyme cannot be eliminated with DEPC. It is better to use microtubes and tipsRnase-free when extracting RNA, and to be extremely careful when separating the phases in extracting traizol.
After surgery and removal of the pancreatic tissue, we immediately transfer the sample into liquid nitrogen and perform all the steps on ice while performing the RNA extraction.
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