I’m always excited to watch curling during the Winter Olympics – great teams from around the world competing and aiming for nothing less than gold. It might look easy as they try to get the stone as close to the button as possible. But it is really much more difficult, because there are so many factors that influence the outcome.
I can easily relate the curling event back to when I was still working in the lab doing gene expression analysis. In a team with my fellow researchers, we tried to isolate RNA so we could obtain reproducible results that we could use to publish our findings before anyone else. And like curling, isolating RNA might look easy to do, but there is actually a lot to consider and many pitfalls where things can go wrong.
Let’s take a look at the gene expression workflow and consider the crucial factors that will help you get the highly pure RNA you need for your downstream applications like RT-PCR or qPCR. In case you’re a beginner in the gene expression, you should first get familiar with general RNA handling tips and tricks. RNA work can be tricky, and you don’t want to ruin your experiment before you even get started.
Sample collection and stabilization
Once a biological sample is harvested, the RNA expression profile in the cell is altered. Sample collection and handling can cause ex vivo gene induction or down-regulation, which can lead to an increase or decrease in RNA expression levels, respectively.
That’s why to capture the true in vivo gene expression pattern, it is important to stabilize your RNA as soon as you finish harvesting your samples. The classical method is to submerge your sample in liquid nitrogen and store it at –80°C. However, these days there are many alternatives, such as RNAlater or other reagents that allow easier handling and processing at room temperature. Here you can see what happens without proper RNA stabilization.
In the non-stabilized sample, the RNA was degraded very rapidly within 10–15 minutes, while in the stabilized sample, RNA was not degraded. For more information on this topic, check out our dedicated blog post Top 9 FAQs in RNA stabilization.
Sample disruption and homogenization
For sample disruption and homogenization it is important to know that these are actually two distinct steps. Disruption helps release all of the RNA in the sample, while homogenization reduces the viscosity of the cell lysates. To develop a suitable sample disruption method that meets your own requirements, you should consider these factors:
- How to best collect and harvest your sample
- The characteristics of your target molecule
- Your required sample throughput
This will help you decide which disruption or homogenization method – chemical, mechanical or a combination of the two – will be most effective for preparing your final homogenate. Agents for chemical disruption may include surfactants (such as detergents), chaotropes or enzymes like proteases, cellulases or proteinase K. Mechanical disruption methods may include ultrasonication, grinding, beating or shearing.
Read more about choosing the right sample disruption method for optimal RNA isolation here.
RNA isolation can be performed in several different ways. Let’s compare the two most commonly used methods: precipitation and the spin-column based method.
Precipitation – also called phase separation – is a phenol-chloroform based method in which mixtures of molecules are separated based on their differential solubilities in two immiscible liquids. Although it is scalable and cheap, the method has a number of downsides: longer handling and incubation times, lack of standardization, high risk of human error, use of harmful reagents and difficult waste disposal. Plus, several rounds of precipitation are often required to obtain a decent purity, so there is a high risk of losing the RNA pellet, something you don’t want and can’t afford.
Spin columns, on the other hand, are easy to work with, are available in different sizes to accommodate various sample amounts and there’s no risk of losing your pellet. They are not only much faster, but they are also more effective at removing genomic DNA (gDNA) than the precipitation method. For this experiment, RNA was isolated from various amounts of RNAlater stabilized rat kidney using the RNeasy Plus Universal Mini Kit, or was precipitated using a precipitation reagent from Supplier AII. RNA eluates were analyzed by real-time RT-PCR, with and without reverse transcription, using the QuantiFast Probe RT-PCR Kit and the Rotor-Gene Q instrument.
An added bonus of the spin-column method is that more than 75 QIAGEN mini spin-column kits are automatable on the QIAcube instrument for various applications including RNA purification from a wide range of sample types. This makes the QIAcube instrument, which can process up to 12 samples at a time, the most dependable lab companion for many scientists, delivering reproducible results time after time without the risk of cross-contamination, as shown in this application note.
Don’t forget about quality control
Reliable results and accurate quantification in downstream assays depend on the quality of the RNA sample. This is why RNA quality control (QC) should be an integral part of every gene expression workflow and should assess three key parameters:
- Quantity – Is there enough RNA to assay?
- Purity – Is the RNA free of contaminants?
- Integrity – Is the RNA degraded?
It’s important that you select appropriate methods for your quality control steps. RNA quantity is commonly assessed by fluorescence measurements in the presence of RNA-binding dyes or by spectral absorbance measurements. While impurities like proteins or chemicals can be detected by spectral absorbance, contaminants such as gDNA in an RNA sample cannot.
The QIAxpert System is our next-generation UV/vis spectrophotometer that not only provides quick and easy concentration measurements but also profiles contaminants within samples. The QIAxpert applies smart analysis algorithms to unmix the spectra and fit reference sample and buffer components to correctly discriminate between DNA, RNA and impurities.
Sample integrity and potential degradation are usually assessed by checking the sample’s size distribution by electrophoretic separation. The QIAxcel Advanced System is our automated capillary electrophoresis system that replaces labor-intensive, manual gel analysis. The system provides an RNA Integrity Score (RIS), which is an objective quality measurement of the analyzed samples that allows easy interpretation of sample integrity. The system can also be used for sample quantification. Based on the migration time and signal intensity, the software can calculate the size and concentration of bands or smears.
When it comes to sample QC, one single technology cannot assess all of the critical parameters. But if you use QIAxpert alongside the QIAxcel Advanced, you can confidently check off all the QC boxes below. Visit our sample Quality Control resource to learn more and find out how to build this important step into your own work.