Effective preparation of RNA is a fundamental technique that is required for a wide variety of exciting and information-rich analysis techniques including next-generation sequencing, reverse transcription qPCR (RT-qPCR), northern blot analysis, and cRNA production. The fidelity of transcriptome representation, and the quality and quantity of recovered RNA, will significantly impact the resulting analysis.
A variety of factors during sample collection, processing, and storage can negatively impact results and should be considered. These factors broadly fall into three categories:
- Stabilization of a sample before processing
- Recovery of RNA during manipulations
- Exposure of unprotected samples to RNases
This article highlights laboratory best practices for RNA preparation and describes 10 ways to improve RNA purification.
- What type(s) of RNA do you need to isolate?
- How to prepare your area for RNA isolation
- How to collect samples and store them before RNA Isolation
- Choosing the right RNA isolation methods/kits
- DNAse - do I have to?
- How much RNA will I get from…?
- Storage of purified RNA
- Testing RNA for quality control
- Know your downstream application
- Bypass the need to isolate RNA by using a cell lysate method
RNA can range in size from 20 nucleotides (nt) upwards of 8 kilobase (kb). RNA isolation procedures require specialized modifications if specific or multiple types/sizes of RNA are desired from the target sample. Thus, it is important to choose the best RNA isolation method for your desired RNA target because not all RNA isolation kits are the same. This link contains a description of the different types of RNA and the associated RNA isolation kits that are recommend for optimal results.
A summary of each types can be found below:
- MicroRNAs (miRNAs) - 18-22 nt small noncoding RNA that function in RNA silencing and post-transcriptional regulation of gene expression
- Small non-coding RNAs (e.g. pre-miRNA, SnoRNA or piRNA) - 20-200 nt long and involved in many cellular processes
- Messenger RNA (mRNA) - >200 nt; transcribe DNA information into the amino acid sequences that make up protein products
- Transfer RNA (tRNA) - 70-90 nt; carry amino acids to the growing peptide chain during protein translation
- Ribosomal RNA or rRNA (e.g. 5S, 18S, and 28S) - >200 nt; RNA component of the ribosome
- Total RNA = all the RNA types together in one eluate
To isolate intact, high-quality RNA, it is essential that RNases are not introduced into RNA preparations once they are no longer protected by strong protein denaturants such as a chaotropic lysis solution or phenol derivatives. RNases are found almost everywhere, and it is essential that any item that could contact the purified RNA be RNase-free. All surfaces, including pipettors, benchtops, glassware, and gel equipment, should be decontaminated with a surface decontamination solution like RNaseZap RNase Decontamination solution or RNaseZap RNase Decontamination Wipes. RNase-free tips, tubes, and solutions should always be used and gloves should be changed frequently.
Endogenous RNases must be inactivated immediately upon tissue harvesting and cell death to prevent RNA degradation.
There are 3 effective methods to accomplish this:
- Thoroughly homogenize samples immediately after harvesting in a chaotropic-based cell lysis solution (e.g., containing guanidinium) like the lysis buffer available in the PureLink RNA mini kit or TRIzol™ Reagent.
- Flash freeze samples in liquid nitrogen. Ensure that tissue pieces are small enough to freeze almost immediately upon immersion in liquid nitrogen to prevent RNA degradation.
- Place samples in RNAlater Tissue Collection: RNA Stabilization Solution, an aqueous, nontoxic collection reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue and cell samples. It is essential that tissue samples be in thin pieces (0.5 cm) so that the RNAlater can quickly permeate the tissue before RNases destroy the RNA.
The wide variety of RNA isolation methods available can make it difficult to decide which one to use. The easiest and safest methods available are column-based methods like the PureLink RNA Mini Kit for mid to low throughput or PureLink Pro 96 Kit for high throughput sample processing needs. Due to ease of handling, these procedures are ideal for working with multiple samples. The MagMAX mirVana Total RNA isolation kit utilizes a paramagnetic particle approach, which is easy to automate on magnetic particle handlers and ideal for processing higher throughput sample needs.
When working with difficult tissues, for example ones that are high in nucleases (pancreas) or fat (brain and adipose tissue), a more rigorous, phenol-based RNA isolation method using TRIzol Reagent is recommended.
- PureLink RNA mini kit – Best and easiest method for most sample types
- MagMAX mirVana total RNA isolation kit – ideal for automated high throughput RNA isolation needs
- TRIzol Reagent – ideal for difficult samples high in DNase or high in lipid content
The PureLink RNA Mini Kit columns are highly efficient for isolating high quality total RNA while removing the majority of genomic DNA. In general, most applications requiring RNA from animal tissue or mammalian cell lines do not require additional DNase treatment. However, some applications such as gene expression analysis by qRT-PCR without intron-spanning primers or working with samples from organisms with very small or no introns may require more complete removal of residual contaminating DNA. The PureLink DNase Set allows for convenient on-column digestion of DNA during the RNA isolation protocol. Treating with DNase while “on-column” is easier and allows higher RNA recovery than treating with DNase after the RNA has been isolated. The PureLink DNase Set can also be used to remove residual DNA from RNA that has been previously purified. Both the “on-column” and post-RNA purification workflows are options available with the PureLink DNase Set.
Some tissues contain abundant RNA content and some contain lower RNA content depending on tissue structure and physiological state. Good sample prep experimental design will prevent a number of sample prep pitfalls such as 1) lower than expected RNA yields from processing insufficient starting tissue amounts, 2) poor quality and/or purity of RNA due to overloading of RNA columns or beads and 3) dilute RNA concentrations if larger than necessary volumes of RNA elution solution are used during isolation. Knowing how much tissue to process before you perform your RNA preparation method will ensure that you isolate sufficient amount of RNA with the expected purity for your applications and purpose.
The chart below serves as a quick check for tissues sample types and their respective RNA yields.
The PureLink RNA mini kit and the MagMAX mirVana Total RNA isolation kits come with RNase-free elution solutions that are optimized for storage of purified RNA. Alternatively, for other RNA isolation methods, such as TRIzol extraction and ethanol precipitation, RNA pellets can be re-suspended in specialized storage solutions such as THE RNA Storage Solution which is a certified RNase-free buffer that minimizes base hydrolysis of RNA.
For short-term storage, purified RNA can be stored at –20°C. However, we recommend storing RNA at –80°C in single-use aliquots to prevent damage to the RNA from multiple freeze-thaw events and help to prevent accidental RNase contamination.
- Store RNA at -20°C for short term storage
- Store RNA at -80°C for long term storage
- Aliquot RNA into several tubes to minimize freeze-thaw and reduce accidental RNase contamination
RNA analysis applications require a defined range of RNA input for the reaction to proceed optimally. Too much or too little RNA may result in failed or false data and misinterpreted results.
How-to measure RNA quality and quantity:
- UV Spectroscopy – The traditional method for assessing RNA concentration and purity. The UV absorbance of a diluted RNA sample is measured at both 260nm and 280nm wavelengths and the nucleic acid concentration calculated from these measurements using the Beer-Lambert law which predicts a linear change in absorbance with concentration. (For more information click on the link: RNA quantitation is an important and necessary step prior to most RNA analysis methods). UV spectroscopy is a simple measurement method and spectrophotometers are readily available in most laboratories. The Thermo Scientific NanoDrop One/OneC microvolume UV-Vis Spectrophotometer has a further benefit as it enables RNA quantitation with only 1-2 µL of sample (traditional spectrophotometers require a much larger volume).
- Qubit Fluorometers – Fluorometric methods are another option to measure nucleic acid using specialized fluorescent dyes. With the Qubit Fluorometer instruments, like Qubit 4 Fluorometer, RNA quantity, integrity and quality can be rapidly measured using highly sensitive Qubit assays even in RNA samples with very low concentration.
- Capillary electrophoresis analysis – RNA quantitation and quality determination based on traditional gel electrophoresis transferred to a microfluidics chip format. These systems provide automated sizing in addition to quantity and quality. RNA integrity number (RIN) can be obtained to assess the quality of the isolated RNA.
Standard acceptable measurements for RNA Quality
- A260/A280 ratio indicates the level of protein contamination. Acceptable ratio for RNA is 1.8-2.0
- RNA Integrity Number (RIN) – indicates the overall “intactness” of RNA. Ideally, RNA analysis techniques recommend using RNA samples with a minimum RIN value of 7. However, some molecular techniques (e.g. qRT-PCR) can tolerate samples with RIN values as low as 2.
RNA can be analyzed via a myriad of applications such as qRT-PCR, microarray, total RNA sequencing, amplicon based/targeted sequencing among others. It is best practice to know the RNA input requirements before RNA purification so you choose the right method and save time and sample.
Other things to consider:
- Do you need to remove any/all residual contaminating DNA? If yes, on-column DNase digestion with Purelink DNase set is easiest and more efficient way to remove DNA
- When analyzing with qRT-PCR, are the primers designed to a single exon gene? If yes, on-column DNase digestion with Purelink DNase set is easiest and more efficient way to remove DNA
- Always Include a “-RT” control for each RNA sample to confirm that RNA is amplified rather than residual DNA
Mastering the complexities of preparing, isolating and quantifying RNA will enhance your ability to understand molecular biology. However, there are certain analytical situations that might allow one to bypass the need to isolate RNA all-together and instead make use of a simpler and faster cell lysate method, saving both time and effort. Cells-to-Ct is one such option. An innovative lysate-based sample preparation method, it enables users to directly lyse from 10 to 100,000 cells in their tissue culture plates. After the short 5 minute lysis step and 2 minute stop reaction step, the cell lysates can be used directly in downstream reverse transcriptase (RT) and qPCR reactions. High throughput and automation friendly, this method allows users to process 96 samples in less than 10 minutes without the need for RNA columns or magnetic beads, centrifugation, heating or expensive sample prep automation systems.
For Research Use Only. Not intended for human or animal therapeutic or diagnostic use.