Have you faced challenges with your RNA workflow? You are not the only one that has been struggling with easily easily degradable RNA.
Working with RNA can be a tricky thing…it falls apart easily, and RNases (enzymes that degrade RNA) are ubiquitous. Successfully isolating RNA and maintaining its integrity is critical, especially when sensitive downstream applications are used (e.g., RNA-Seq).
Good techniques for RNA handling are simple to employ but crucial for success. All RNA purification and handling should take place in an RNase-free, RNA-only zone of the lab. Segregating RNA work from protein and DNA purification and handling will help minimize the potential for RNase contamination and help keep your RNA intact. Only buffer and water stocks treated to be RNase-free should be kept in the RNA area of the lab, and gloves should be worn at all times to prevent accidental contamination. Tools and equipment such as pipets, tips, and centrifuges should be designated for use only in the RNA zone as well. The location of the RNA zone in the lab is also important. Keeping traffic to a minimum and moving the RNA zone away from doors, windows, and vents can also help minimize contamination.
Promega has technical service scientists who are working hands on to find you the answers and help that you need to succeed in your experiments. They develop top of the art products and back them up with tips and tricks for entire RNA workflows and all the other technologies Promega offers. Below you can find some key points to consider when working with RNA.
RNA is precious and degrades easily as the environment is filled with RNA-degrading enzymes RNAses. Choose your method of isolation carefully and once you have the RNA, protect it properly with RNAse inhibitors until used in experiments. RNase inhibitor helps safeguard your samples from RNase degradation by binding to any RNases that may have been introduced into your sample and prevent them from cutting the RNA present. Promega has RNA kits for fast and efficient RNA isolation and RNAsin to keep your RNA safe after extraction.
Best measurement of RNA after purification is with fluorophoric dyes. They bind to nucleic acid and hence provide most accurate means to get concentration and amount of RNA correct.
Did you know that often nucleic acid purification techniques can result in inhibitors in your PCR-reaction and choosing the optimal purification might be essential for the perfect PCR? Especially so with RT-PCR. How do you know if you have inhibitors in your eluted RNA? And if there is inhibition, is it in the RT-reaction or in the qPCR-reaction? The shape of slope and differenced in Cq-values tells you if one of the two major reactions is inhibited or if there is something in your RNA that interferes with the fluorescent signal needed to record the real time amplification events.
Read more of avoiding inhibitory problems and how to identify them in this blog: https://www.promegaconnections.com/dealing-with-pcr-inhibitors/
The enzyme is not the only thing making your PCR work – a lot can be made with choosing the optimal reaction conditions and especially buffering of the reaction. https://www.promegaconnections.com/optimizing-pcr-one-scientists-not-so-fond-memories/
The time you spend optimizing the reaction is what you save when having robust data from your experiments. Promega has PCR satisfaction guarantee: if we can’t make your PCR work, you get your money back.
Let us help you
Would you benefit from having a great RNA workflow: proper extraction method, a robust enzyme and robust buffer knowhow for all your reactions? If yes, please let us help you find the perfect PCR-system for your needs! Promega has technical services for guiding you further and even a fully equipped applications lab to optimize the PCR or the entire workflow for your needs. Let us help you!
Heidi Bondén, puh. 050 356 3565 heidi.bonden(at)labnet.fi
Pia Lindström, puh. 040 509 8744 pia.lindstrom(at)labnet.fi