Hello everyone,
What is the best or a useful way to eliminate possible RNase contamination from lab equipment including plasticware and glassware when extracting RNA from FFPE tissues?
Ps: I'll be working with the RNeasy FFPE kit from Qiagen.
Thank you.
Hello Ayman Reffai
Please refer below:
Plasticware
Soak the plasticware in 0.1N sodium hydroxide in 0.1% EDTA in water for 2 hours at 37 degree C and then rinse thoroughly with 0.1% (v/v)DEPC-treated water. Then heat the plasticware at 100 degree C for 15 minutes in an autoclave.
Glassware
Soak the glassware in freshly prepared 0.1%(v/v) DEPC in water for one hour. Then drain the glassware and autoclave. Autoclaving will destroy the untreated DEPC which could react with RNA.
Equipment
Clean the equipment with a detergent solution like 1%SDS, rinse thoroughly with water, and then rinse with 95% ethanol and dry. Soak the equipment in 3% hydrogen peroxide for 10 minutes at room temperature. Then rinse the equipment thoroughly with DEPC-treated water (0.1% DEPC) followed by autoclave water.
I hope this is helpful.
Good Luck.
Use mask and avoid talking when doing isolation.
For plastic wares its better to treat with DEPC water fallowed by double autoclave.
Pipettes and working bench clean with RNase Zap.
Always use ice when you are performing manual method of isolation.
Use mask and avoid talking when doing isokation.
Hi Ayman, kindly have a thorough look into one of my earlier answers few years back to get more insights and tips to solve this problem, more precisely a nightmare for researchers working with RNA.
https://www.researchgate.net/post/What-is-the-protocol-of-using-DEPC-in-RNA-extraction
Simultaneously I would highly recommend to use always "RNAse inhibitors" during your cDNA (RT) step to avail maximum protection for your RNA content in each reaction mix. You can opt for the following one here or some times it comes with certain cDNA kits as well. You will notice quite fine cDNA quality by using the RNAse inhibitors.
https://www.thermofisher.com/order/catalog/product/N8080119
Regards....
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