In Vitro Transcription: Common Causes of Reaction Failure

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A widely used molecular biology technique, in vitro transcription uses bacteriophage DNA-dependent RNA polymerases to synthesize template-directed RNA molecules. Enzymes like bacteriophage SP6, T3 and T7 RNA polymerases are used to produce synthetic RNA transcripts, which can be used as hybridization probes, as templates for in vitro translation applications, or in structural studies (X-ray crystallography and NMR). Synthesized RNA transcripts are also used for studying cellular RNA functionality in processes such as splicing, RNA processing, intracellular transport, viral infectivity and translation.

Problems in the transcription reaction can result in complete failure (i.e., no transcript generated) or in transcripts that are the incorrect size (i.e., shorter or longer than expected). Below is a discussion of the most common causes of in vitro transcription problems.

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Lost in Translation? Tips for Preparing RNA for in vitro Translation Experiments

A protein chain being produced from a ribosome.
A protein chain being produced from a ribosome.

Synthesizing proteins in vitro through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function.  If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free vs. long growth times and extraction steps after an initial optimization for protein synthesized in larger scale).  There are many systems out there, and knowing which to use can sometimes be difficult.  Many kits include components that combine transcription and translation in one-step, eliminating the need to provide your own RNA.  But when you want to make your own RNA templates to add to lysates, then there are additional concerns.

Many people don’t want to work with RNA since the common lab lore suggests it’s a finicky molecule, and for good reason.  Extracting it requires the utmost care in technique and elimination of nucleases.  Failing to do so results in degradation of the molecule, and so with it your experiments (see our recent blog by Terri Sundquist on tips for isolating RNA with ease).  Preparing RNA for cell-free expression is subject to the same concerns as extracted RNA, but with the proper care is not that much more of a challenge than using a DNA template.

The first step for using cell-free expression systems with RNA templates is to make the RNA.  Here are some tips that will ensure success. Continue reading “Lost in Translation? Tips for Preparing RNA for in vitro Translation Experiments”

How to Isolate RNA like a Pro

Ribbon diagram of RNA’s biggest threat: a ribonuclease
Ribbon diagram of RNA’s biggest threat: a ribonuclease

Back in graduate school, I purified a lot of RNA, and after a while, I became fairly successful at it. My yields were good, and the RNA was intact. However, many of my early attempts at RNA isolation yielded degraded RNA that did not work well in many downstream applications. In my case, successfully isolating high-quality RNA required practice. During my trials and tribulations, I learned a lot of tricks and tips about how to obtain high-quality RNA. Here I share some of these tricks to help you speed through that “practice makes perfect” phase so that you can isolate RNA like a pro.

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Don’t Let Ribonucleases Ruin Your Week(end): Establish a Ribonuclease-free Environment

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My very first job in science was in a lab that worked exclusively with RNA, and it was only after I moved on to a different job that I learned just how much different the world of DNA research is from that of RNA. When working with DNA, for example, you rarely if ever have the sample you have labored over reduced to a fuzzy blur at the bottom of a gel because it has been degraded beyond rescue. With RNA, unfortunately, this happens all too frequently. In fact, a labmate of mine once put up a poll on the door to our lab asking if it was better to discover that your RNA sample was degraded on a Monday or a Friday.

The culprits in this scenario are Ribonucleases (RNases). They are everywhere. They are incredibly stable and difficult to inactivate. And, if you work with RNA, they are your enemy. Take heart though, they can be defeated if you follow some pretty simple steps.

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Working with RNA

Set up a lab RNA Zone

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.

Using an RNase inhibitorcan also help safeguard your samples from RNase degradation. These inhibitors can bind to any RNases that may have been introduced into your sample and prevent them from cutting the RNA present.

Water and buffer stocks can be a source of RNase contamination. Several stocks from an RNase-free zone in an academic lab showed RNase activity. Recombinant RNasin® inhibitor protected all RNA samples from degradation.