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The Phobia of RNases
My first experience of troubleshooting in vitro transcription came when I was synthesizing RNA In-Situ Probes for the first time. A lab mate ominously warned me that I had just returned to lab after a bout of flu and that meant I’m a walking talking factory of RNases. I went ahead anyway and sure enough, I had no RNA following the in vitro transcription step. I tried once more, and was extra careful this time. I literally bathed myself, my work bench and the pipettes in RNase zap! I even wore a face mask, so I wouldn’t exhale RNases into my samples. Didn’t work.
I gave myself a week (to become completely RNase free!) and got back to work (and after another week of systemic troubleshooting) to find out that my RNA polymerase was denatured. So that you don’t also have to go through the pain of weeks of failed in vitro transcriptions, I’ve put together a checklist for troubleshooting in vitro transcriptions.
Troubleshooting in vitro Transcription
There can be several reasons why your in vitro transcription does not work and RNase contamination is just one of them. Here, I suggest a few tricks to work your way through when troubleshooting in vitro transcription, without blowing up your paranoid head!
- Make sure you work RNase free. RNA is delicate and the threat of RNases is everywhere. So, in order to ensure success, you really need to make sure that you are working as RNase free as possible. You can find helpful hints in our ‘10 ways to work RNAse free’ article.
- Be careful, but be quick. As mentioned above the threat of RNases is everywhere. Carry out the steps as fast as you can, minimizing exposure to the environment. RNAses are omnipresent! But remember there is a balance between speed and accuracy, so don’t move so quickly that you make errors that cause your experiment to fail.
- Work on ice. RNAases, like most enzymes, have sluggish activity at low temperatures. This means you can slow down their ability to destroy your RNA (and your experiment) by keeping everything cool, so bring out those ice troughs to set up the reactions. Cold block tube stands are a good resource to invest in. You can place them on the ice in the ice troughs. The reagent tubes and the reaction tubes can be made to sit on them. This means that the tubes and their contents are being kept cool while at a safe distance away from the ice that could potentially be a contamination risk.
- Know your Reagents. The RNA polymerase is hypersensitive and demands its fair share of respect. It is a protein and can get denatured when subjected to drastic temperature changes. It is a good idea to aliquot and store it, thereby minimizing too many freeze-thaw cycles. I recommend storing the stock at -80ºC, and storing 5µL aliquots at -20 ºC in Mini-coolers.
- Include an RNAase inhibitor in the reaction mixture. There are lots of different inhibitors available so do look around or even ask your colleagues what they use (and if they have some you could use). Personally, I have always used RiboLock RI.
- Watch for turbidity. The transcription mixture should begin to turn turbid after around 15 minutes of setting up the reaction. This turbidity, or viscosity, is essentially your RNA being transcribed and then eventually precipitating out of the reaction mixture. This means that if you have waited for an hour and your solution is still bright and clear, you can throw it away.
- Incubate for the right length of time. A 3- to 6-hour incubation of a 50 µl solution at 42º C yields a minimum of around 30 µg of ssRNA (yes, 42º C instead of the usual 37º C). The incubation time depends on the concentration and amount of ssRNA you want. Beyond 6 hours, I haven’t seen much of a drastic increase in the yield. Understandably, with the increase in RNA concentration in the reaction mixture, the pH and buffer composition are disturbed, slowing down transcription.
To incubate, add little water to a heat block and place the tube in it. The heat block allows for a tight control of temperature. The water cushion around the tube works like a double boiler system. Water baths are a slightly more contamination prone alternative.
Storing Your RNA
You have your ssRNA and can now pellet it and check for concentration as well as check for quality. The RNA can be stored in RNase free water at -80 ºC, such that the concentration is greater than 1 µg/µl. For batches that you are going to use immediately, make 20 µl 1:10 dilution aliquots of the above solution and store it safely at -20 ºC.
Using your RNA
For RNA In Situ experiments, in case your transcript is more than 300 bp long, you’d have to further hydrolyze it so that the fragments ease into the cells conveniently and bind with your target RNA. Since this is an additional step, there is so much more scope for you to lose out on the yield. It is always safer to completely do away with this step and to start with designing a probe that’s around 200-300bp long.
I hope you’ve found this article useful for troubleshooting in vitro transcription. Good luck!