Welcome back to our RNA-seq series! In Part 2, we take a look at RNA quality considerations for RNA-seq.
As outlined in Part 1, library preparation is a pivotal part of the RNA-seq workflow. Although the exact protocol will vary according to RNA-seq platform technology (more about this in Part 3), the overall goal is the same: to create a library of complementary DNA fragments that represent the originating RNA sample as accurately as possible and provide information about the RNA features of interest, including information about antisense transcripts, alternative splice variants, miRNAs and other non-coding RNAs, low-abundance transcripts and more.
There are a plethora of commercial kits available that make the job of library preparation quite simple and give good yields, but the outcome of any of these kits is entirely dependent on the quality of the starting material i.e. the RNA quality.
What is RNA Quality?
When we talk about RNA quality for RNA-seq or indeed any RNA-based application, we are referring to both RNA integrity and purity.
RNA Integrity and Purity
RNA integrity refers to overall intactness of the RNA subunits. Since RNA is a chemically unstable molecule, it is inherently susceptible to RNase degradation so care must be taken at all stages during any RNA-based experiment to prevent or keep degradation to an absolute minimum. Starting out an RNA-seq experiment with degraded DNA will result in low library yields or worse – complete failure to generate libraries. Even if you manage to generate a library, your data may not be reliable since transcripts will likely be differentially degraded.
Even when the integrity is good, RNA can suffer from a number of impurities, including organic solvents and salts left over from extraction (e.g., phenol, chloroform), genomic DNA (gDNA), nucleases as well as other proteases that can make it through the extraction process. All of these impurities pose potential problems with downstream processing steps e.g., by inhibiting or digesting the enzymes that carry out reverse transcription and library synthesis, or by skewing the data e.g., sequencing reads originating from contaminating gDNA.
In the next sections, we share advice to help you maximise RNA integrity and purity as well as straightforward ways to assess RNA quality.
How to Maximise RNA Integrity
1. Fresh is Best
If possible, aim to work with freshly isolated samples. This way you avoid the degradation that may occur during storage and freeze-thaw cycles, boosting your chances of getting a true molecular snapshot of your sample at the time of collection.
2. Zap Those RNases
Whether you are fortunate enough to have access to fresh samples or not, you will need to take care when working with samples destined for RNA isolation. Some common practices include:
- Working double-gloved on ice (ice should deactivate RNases)
- Using dedicated RNA pipettes (in an attempt to avoid DNA contamination)
- Using pipette tips with filters to avoid RNase or other contaminants entering through the pipette shaft
- Working in designated RNase-free zones
- Cleaning all surfaces with commercial or homemade RNase-deactivating solutions
3. Preserve Your Sample for a True Snapshot!
While the steps above are certainly good practice that should help to maximise RNA integrity, you should also consider the biological factors that could skew the RNA picture (i.e. the molecular snapshot) you want to examine.
For example, if you want to profile the metatranscriptome in a mouse intestine in response to different diets, you will probably use stool samples as your starting material. However, scooping the poop won’t freeze that sample in time. The bacteria present in the gut contents will continue to divide and metabolise after sample collection, and some transcripts may even be lost during this process unless you freeze bacterial and enzymatic activity in some way. We recommend doing this with a nucleic acid stabiliser such as DNA/RNA ShieldTM rather than freezing, to avoid freeze-thaw cycles.
How to Maximise RNA Purity
1. Choose Your Protocol Wisely!
Nowadays we are spoilt for choice when it comes to isolation kits, but they are not all equal. If you are using a spin column-based kit, it’s worth noting whether the column is optimised to not retain buffer traces during washing steps. Kits that eliminate buffer retention will help to minimise impurities that may otherwise make it through to your isolated sample. Sometimes it also helps to add additional wash steps if you experience impurities during quality control (more about this below).
2. Post-isolation Clean Up
If using highly optimised spin columns and adding extra wash steps is not enough to eliminate impurities, you do have some other options. You could try a post-isolation clean up kit to help shift impurities such as TRIzol, chloroform and other organic solvents. These cleanup kits can also be used to remove unused components of enzymatic reactions e.g. if you choose to perform a post-isolation DNase digestion.
Alternatively, and particularly if working with complex or secondary metabolite-rich samples such as soil, plants or fungi, you may wish to use a PCR inhibitor removal kit to remove polyphenolic compounds, pigments and other impurities that may wreak havoc for downstream enzymatic reactions.
3. DNase Treatment
No matter how effective your isolation kit or protocol may be, a certain amount of gDNA is likely to end up in your isolated RNA samples. Some commercial kits include a DNase treatment step that is carried out during the isolation protocol (on-column), while others include this step post-isolation. If you are using a non-kit-based traditional method, we recommend that you include a post-isolation DNase treatment step before proceeding with library preparation.
Quality Control – How?
You can quality control your RNA samples by assessing integrity and purity using the following approaches:
1. Gel or Capillary Electrophoresis
Good ‘ol fashioned agarose gel electrophoresis is still a popular method to visualise RNA integrity. Here, you resolve a portion of each sample on a denaturing agarose gel and stain with ethidium bromide or another nucleic acid-binding stain that is visible under a UV lamp.
Intact RNA will appear as sharp ribosomal RNA (rRNA) bands. The exact subunit sizes will depend on the organism you are working with, but if your RNA is intact you should clearly see a large and small subunit in an intensity ratio of approximately 2:1. Partially degraded RNA will appear as a smear along the lane, the rRNA bands won’t be sharp and the intensity ratio will be off. Low molecular weight smears indicate completely degraded RNA.
Although gels provide a useful visual indication of integrity, they don’t provide any quantitative measure of intactness and it is impossible to accurately and consistently quantify the subunit ratios by eye.
To get quantitative information about RNA integrity, you may consider using capillary electrophoresis with automated data analysis e.g., Agilent Bioanalyzer or Tape Station or similar. These platforms quantify the intensity ratio between the large and small rRNA subunits and provide this as an RNA Integrity Number (RIN). There is no universal RIN threshold for RNA-seq workflows but RIN values below 7 are generally not accepted. An additional benefit of capillary electrophoresis systems is that they also provide information about the concentration of RNA present in the samples.
Traditional spectrophotometers and nanophotometers are widely used to provide information about RNA concentration and purity. Concentration is assessed by measuring the absorbance at A260 nm, and purity can be assessed by comparing the following absorbance ratios:
- 260/280 nm ratio: A ratio of 2 is usually interpreted as pure for RNA. Lower ratios may indicate the presence of proteins or contaminants such as TRizol, phenol and others that absorb strongly at or near 280 nm.
- 260/230 nm ratio: A ratio of 2-2.2 is generally accepted as pure RNA. Lower ratios indicate the presence of contaminants such as EDTA, carbohydrates and phenol, TRizol and others that absorb near 230 nm.
Although widely used, spectrophotometric methods are limited by the fact that will measure everything that absorbs at a given wavelength. This means that they cannot distinguish between RNA, ssDNA, dsDNA and single nucleotides, meaning that potential gDNA present in your sample will be included in your RNA quantification. Where this is a concern, fluorometric detection kits are recommended. The use of RNA-specific binding dyes ensures that you only measure RNA.
RNA Quality In Practice
Each of the quality control methods described above has its merits, and you will probably use a combination of these to build a trustworthy picture of the integrity and purity of your samples. If you are unsure about which approaches are best for you, or if you want to take measures to improve the quality of your RNA samples, do get in touch with us by writing an email to email@example.com or find your local representative here. We are always happy to help!