Working with RNA doesn’t have to be a nightmare

We’re all familiar with the Central Dogma of Molecular Biology: DNA is transcribed into RNA, which is translated into proteins. It’s drilled into our heads from the early days of biology classes, and it’s surprisingly useful when we start exploring in our own research projects. For example, if you’re interested in gene expression, you’ll most likely be working with RNA, specifically mRNA. Messenger RNA (mRNA) is transcribed from DNA and is used by ribosomes as a “template” for a specific protein. The total mRNA in a cell represents all of the genes that are actively being transcribed. So, if you want to know whether or not a gene is being transcribed, RNA purification is a great place to start.

When preparing your RNA samples for a downstream assay, there are several roadblocks and pitfalls that could give you quite a headache. Let’s tackle two of the most common.

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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.

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

Proteinase K: An Enzyme for Everyone

protein expression purification and analysisWe recently posted a blog about Proteinase K, a serine protease that exhibits broad cleavage activity produced by the fungus Tritirachium album Limber. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples. In that previous blog we focused on its use to remove RNase and DNase activities. However, the stability of Proteinase K in urea and SDS and its ability to digest native proteins make it useful for a variety of applications. Here we provide a brief list of peer-reviewed citations that demonstrate the use of proteinase K in DNA and RNA purification, protein digestion in FFPE tissue samples, chromatin precipitation assays, and proteinase K protection assays: Continue reading “Proteinase K: An Enzyme for Everyone”

ProK: An Old ‘Pro’ That is Still In The Game

Proteinase K Ribbon Structure ImageSource=RCSB PDB; StructureID=4b5l; DOI=http://dx.doi.org/10.2210/pdb4b5l/pdb;
Proteinase K Ribbon Structure ImageSource=RCSB PDB; StructureID=4b5l; DOI=http://dx.doi.org/10.2210/pdb4b5l/pdb;
If you enter any molecular lab asking to borrow some Proteinase K, lab members are likely to answer: “I know we have it. Let me see where it is”. Sometimes the enzyme will be found to have expired. The lab may also have struggled with power outages or freezer malfunctions in the past. But the lab still decides to keep the enzyme. One may rightly ask – why do labs hang on to Proteinase K even when it has been stored under sub-standard conditions? Continue reading “ProK: An Old ‘Pro’ That is Still In The Game”

Choosing the Right Reverse Transcriptase for Your Project

There are a lot of choices when it comes to reverse transcriptases.  Choosing the correct one for your cDNA synthesis and RT-PCR project is important.    Here are a few questions that will lead you to right RT for your application: Continue reading “Choosing the Right Reverse Transcriptase for Your Project”

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.

Fixed in the Past, Focus on the Future

“I would do more with my samples, but it’s just not possible…I know there’s probably a wealth of information in there, but there is just no way to get it out…I’ve got blocks of tissue sitting in the lab, experiments I want to run, but no good way to get clean nucleic acids out.”

These are a few of the comments I heard when talking with scientists at the American Society of Human Genetics meeting last week in Montreal. They, and countless other researchers, are sitting on a treasure trove of information that may have been locked away a few months ago, a few years ago, or decades ago. I’m referring to formalin-fixed, paraffin-embedded (FFPE) tissue blocks. It is estimated that there are upwards of 400 million tissue blocks archived globally and scientists are clamoring to find ways to best utilize nucleic acids derived from these tissues in applications like qPCR, microarrays, and next generation sequencing.1  Continue reading “Fixed in the Past, Focus on the Future”