Even the most experienced molecular biologist has screamed their favorite expletive during an RNA extraction. To a non-scientist, it can look like this poor person is losing it, yelling at an innocent tube, filled with clear, nondescript liquid.
But to those scientists who have experienced the many woes of RNA extraction, it’s easy to empathize. Outpourings of anger (also known as RNAnger) are a natural and common side effect of working with RNA. And it’s understandable: Low extraction yield and degraded RNA can happen to the most senior scientists.
Obtaining high-quality RNA from sample types has become a fundamental part of so many protocols, including next-generation sequencing (NGS), quantitative real-time PCR (qRT-PCR), northern blotting, and cDNA synthesis, and many scientists perform it routinely. Low-quality RNA can significantly impact downstream analysis, leading to lost time and money.
It doesn’t have to be this way. Many RNA extraction issues can be avoided and conquered. To empower you on your journey to RNA extraction victory, we have 14 tips to take your RNAnger and transform it into RNAccumen.
Select the Right RNA Isolation Kit
There are a lot of kits and reagents out there for RNA extraction. But certain protocols or formats may be better suited for your goals. Do careful research on each kit or reagent and consider the type of sample you’ll be extracting RNA from, the starting amount of that sample , your downstream analysis method, required throughput, and the type of RNA you’re analyzing.
For example, if you need a high-throughput purification method for total RNA, then you may want to consider the scalable MagMAX™ kits instead of TRIzol RNA extraction. But if you have a small number of samples and want to use a gold-standard method for high-quality RNA, you’ll likely pick TRIzol every time.
Pay Attention to Input Amounts
More isn’t always better. Most RNA isolation kits and reagents provide a recommended number of starting material for a given sample type. Adding more than is recommended can inhibit effective lysis or separation of RNA from other cellular components. For column-based protocols, you may overload the binding capacity of the column, which can reduce overall yields.
Be aware that the RNA contents can vary significantly in different animal tissues: Heart and muscle cells have less overall RNA than spleen and thymus cells, which tend to be nucleic-acid rich. These tissues can also introduce additional confounding factors that may complicate extraction and protocol modifications may be needed to extract high-quality RNA.
Create an RNAse-Free Environment
RNases? Never heard of ‘em, right?
They are the number one enemy of your RNA extraction. They can be tough to inactivate or get rid of. RNases in the RNase A family are stabilized by several disulfide bonds and can retain activity, even after being denatured.1,2
To mitigate the accidental introduction of RNases during your RNA extraction, create an RNase-free benchtop sanctum. This area can be a section of your bench, marked off with tape that is separate from where you do DNA or protein extractions. Keep reagents, consumables, pipettes, and gloves in this area that are dedicated to your RNA extraction work.
Wear Your PPE
You are a prime source of RNases. Your perspiration and skin oil is a rich source for RNase contamination.3,4 For that reason, wearing gloves to prevent contamination from your hands is critically important. And if you happen to use the gloves you’re wearing to touch a surface that could have RNases on it (i.e., doorknob, your face, or other surfaces outside of the RNAse-free zone you created above), be sure to change your gloves. You can also wear your lab coat to make sure that no other part of your skin comes in contact with your samples.
Use RNAse-Free Reagents and Consumables
Aligned with the above point, you can also purchase cleaning products, consumables, and other reagents that can help keep the RNases at bay, ensuring that they don’t come anywhere near your RNA. Surface decontamination solutions can help remove RNases from glassware, benchtops, and pipettes.
You may assume that all tubes and pipette tips are RNase-free out of the box, but many may have had RNases introduced during manufacturing. Be sure that your consumables are certified as RNase-free and that you use filter tips to prevent contamination from your pipette tip or cross-sample contamination.
Run a Pilot Experiment
You may not realize that a potential step exposes your RNA to RNase contamination or that the kit you’re planning to use isn’t suitable for your sample type until you start your RNA extraction. To prevent you from squandering your valuable samples, run a pilot experiment with less valuable samples so that you can work out the kinks in your RNA extraction protocol. This can save on costly rework or having to re-collect samples.
Stabilize Your RNA
The biochemical nature of RNA makes it inherently unstable and some RNAs have short half-lives.5,6 To make sure you aren’t biasing your downstream analysis, you’ll want to quickly disrupt all endogenous RNases as quickly as possible after sample collection. You can do this by lysing cells using TRIzol or lysis buffer from our PureLink™ RNA Mini Kit and freezing samples at -80° C for later processing. You can also extract RNA immediately after resuspension in lysis buffer. Alternatively, flash freezing samples in liquid nitrogen can prevent RNA degradation after sample collection.
If you don’t want to deal with the difficulties of liquid nitrogen, you can use RNA stabilization reagents, such as RNAlater. It inactivates RNases immediately and gives you the flexibility to extract RNA when you have time, without having to worry about RNA degradation.
Add Mechanical or Enzymatic Lysis Steps
FFPE samples, blood samples, and certain microbes are notoriously challenging to extract a sufficient amount of high-quality RNA from and may require additional steps to ensure complete lysis. These can include mechanical lysis using bead-beating or enzymatic pre-treatment using lysozyme or proteinase K. As mentioned earlier, incomplete lysis may cause downstream issues with column-based extraction methods. Before extracting RNA from a challenging sample, carefully review the protocol to see if they include specific protocol steps for challenging samples.
Keep Things Cold
The instability of RNA makes it critical to keep samples cold throughout processing. If you’ve encountered issues with RNases before, consider keeping extraction solutions cold, doing the extraction in a cold room, or using a pre-chilled centrifuge for centrifugation steps.
Remove DNA Contamination
Most RNA isolation kits and reagents do an effective job of removing contaminating genomic or plasmid DNA. But certain downstream quantification methods, such as NanoDrop, or applications, such as qRT-PCR, can detect even minuscule amounts of DNA and complicate analysis.
To rectify this, many column-based purification methods include DNase I, like PureLink™ DNase Set, which can be used for on-column treatment. You can also use DNase I for post-extraction digestion.
Quantify RNA and Run Quality Control
There are several methods for running quality control on your purified RNA and getting accurate quantifications. You can use UV absorbance at wavelengths 260nm (for nucleic acids), 280nm (for contaminating protein), and 230nm (for other contaminants like guanidine isothiocyanate). RNA purity can be estimated by looking at the ratio of A260 / A280 (aim for between 1.8 and 2.2) and A260 / A230 (aim for >1.7).
Fluorometers, such as the Qubit Fluorometer, can also quantify RNA and are more sensitive than UV absorbance-based methods. Microfluidics-based methods are also helpful in quantifying RNA and determining quality. Samples are labeled with a fluorescent dye and electrophoresed through a gel matrix. Different RNA species migrate through the matrix as a function of size and RNA integrity can be estimated by looking at a full trace of fluorescence. The method can detect DNA contaminants or RNA degradation products and formulate them into an RNA Integrity Number (RIN) on a scale of 1 to 10, with 10 indicating the best RNA integrity.
Store Your Purified RNA
Many column- or bead-based kits provide RNase-free elution buffers that can protect the integrity of RNA long-term. For TRIzol RNA extraction, you can resuspend dried pellets in RNase-free water or other special storage solutions. After quantification, aliquot your RNA into single-use tubes so that you can minimize freeze-thaw cycles (which can lead to degradation) or accidental RNase contamination. If you plan to use your RNA in the short term, store it at -20° C. Otherwise, store it at -80° C for longer-term storage.
Automate Your Extraction
Several RNA extraction kits available (including the MagMAX kits mentioned earlier) can be used with automated extraction and purification systems, such as the KingFisher™. Automation helps to avoid one of the major issues with RNA extraction – RNase contamination from human contact. It also enables reproducible RNA purification and reduces the amount of hands-on time by 75%, compared to manual sample preparation.
Rock every step of your RNA extraction
Ask for Help
Some problems are too big to solve by ourselves. If you need additional support and help with RNA extraction, visit our RNA sample collection, protection, and isolation support center. There are guides, basic tutorials, troubleshooting tips, training, and much more to help you navigate the path to successful RNA extraction.
To continue your learning, we have a additional online resources:
This article is for Research Use Only. Not for use in diagnostic procedures.
- Working with RNA: the basics. Thermo Fisher Scientific website: https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FBID%2FTechnical-Notes%2Frnasezap-rnase-decontamination-solution-tech-note.pdf&title=VGVjaCBOb3RlOiBSTmFzZVphcCBSTmFzZSBEZWNvbnRhbWluYXRpb24gU29sdXRpb24uIFdvcmtpbmcgd2l0aCBSTkE6IHRoZSBiYXNpY3M=. Published May 2, 2019. Accessed July 26, 2021.
- Klink TA, Woycechowsky KJ, Taylor KM, Raines RT. Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A. Eur J Biochem. 2000;267:566-572.
- Gupta SK, Haigh BJ, Griffin FJ, Wheeler TT. The mammalian secreted RNases: mechanisms of action in host defence. Innate Immun. 2013;19:86-97.
- Zasloff M. Antimicrobial RNases of human skin. J Invest Dermatol. 2009;129:2091-2093.
- RNA. EMBL-EBI website: https://www.ebi.ac.uk/training/online/courses/biomacromolecular-structures/rna/. Accessed July 28, 2021.
- Sharova LV, Sharov AA, Nedorezov T, Piao Y, Shaik N, Ko MS. Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells. DNA Res. 2009