Working with RNA: Hints and Tips
Working with RNA
An RNase free environment is essential when working with RNA samples. In the laboratory, obtaining full length, high quality RNA can be challenging. There are two main reasons for RNA degradation during RNA analysis. First, RNA by its very structure is inherently weaker than DNA. RNA is made up of ribose units, which have a highly reactive hydroxyl group on C2 that takes part in RNA-mediated enzymatic events. This makes RNA more chemically labile than DNA. RNA is also more prone to heat degradation than DNA.
Secondly, enzymes that degrade RNA, ribonucleases (RNases) are so ubiquitous and hardy; removing them often proves to be nearly impossible. For example, autoclaving a solution containing bacteria will destroy the bacterial cells but not the RNases released from the cells. Furthermore, even trace amounts of RNases are able to degrade RNA. Therefore, it is essential to avoid inadvertently introducing RNases into the RNA sample during or after the isolation procedure.
Sources of RNases
The presence of RNases on human skin surfaces has been well documented. RNase contamination through this source is very easy to acquire and spread if tubes, pipette tips, bench tops, etc. are touched with bare hands.
Dust particles floating in the air often harbor bacteria or mold. The RNases from these microorganisms are deposited wherever the dust settles. This includes lab equipment, open bottles, etc.
If the reagents used for RNA analysis are not certified to be RNase free, there is a good chance that some of the contamination will come from this source. Reagents can also become contaminated in the lab itself if proper care is not taken.
RNase contamination can come from the samples themselves as tissues and cells contain endogenous RNases.
How to Maintain an RNase-Free Environment
- Disposable sterile plasticware
- Non-disposable plasticware
- Good quality reagents
- DEPC-treated water
- RNase inhibitors
- Aseptic techniques
- Decontamination techniques
Always wear sterile disposable gloves when handling reagents and RNA samples. It is important to remember that once the gloves have touched equipment in the lab such as centrifuges, pipettes and door handles, they are no longer RNase free. Change gloves frequently as the protocol progresses from crude extract to more purified material.
Disposable plasticware greatly reduces the possibility of contaminating your samples and are typically RNase-free (check with supplier). In the event of a contamination, it also minimizes spread of the contamination. The use of disposable tips, tubes, etc. is therefore highly recommended.
Non-disposable plasticware should be treated before use to ensure that it is RNase-free. Plasticware should be thoroughly rinsed with 0.1M NaOH, 1mM EDTA followed by RNase-free water. Alternatively, chloroform-resistant plasticware can be rinsed with chloroform to inactivate RNases.
Always ensure that all reagents and chemicals purchased commercially are guaranteed to be RNase free. Testing each batch before use may be a prudent step.
Use DEPC-treated water instead of regular PCR grade water. DEPC inactivates RNase by histidine modification of the bases. If DEPC-treated water is made in-house, always remember to autoclave before use to degrade the DEPC.
The use of RNase inhibitors such as RiboSafe RNase Inhibitor is highly recommended with samples containing endogenous RNase.
Always use proper microbiological aseptic techniques when working with RNA.
Heat-proof glassware should be cleaned with a detergent and rinsed thoroughly prior to being baked at 180°C for several hours to inactive RNases. Autoclaving alone will not inactivate many RNases. Polycarbonate or polystyrene materials can be decontaminated by soaking in 3% hydrogen peroxide for 15 minutes, followed by thorough rinsing with RNase-free water.
Correct storage of RNA is also very important to avoid RNA degradation. In the short-term, RNA can be divided into small aliquots and stored in RNase-free water or TE buffer at -80ºC for 1 year without degradation (freeze/thawing more than once is not recommended). For long-term storage RNA samples may be stored as ethanol precipitates at -20°C. However, when dissolved in ethanol, RNA is not dispersed evenly in the solution and cannot be used directly in quantitative experiments. Instead, precipitates should be pelleted and redissolved in an aqueous buffer before pipetting. When working with RNA on the bench, keep the RNA aliquot on ice and the lid closed when performing other steps.
Extraction and preparation of RNA
There are number of RNA preparation technologies available, the two most commonly used are organic extraction and column purification.
Organic extraction, such as the use of Triazol, is considered the gold standard for RNA preparation, where the sample is homogenized in a phenol based solution that removes any RNase activity and then centrifuged to separate the sample into three phases: a lower organic phase, a middle phase that contains denatured proteins and genomic DNA and an upper aqueous phase that contains RNA. The upper aqueous phase is recovered and RNA is collected by alcohol precipitation and rehydration. The main drawbacks of this process are firstly it is laborious and manually and secondly there is the use and associated waste of chlorinated organic reagents.
Column based formats, such as the ISOLATE II RNA Mini Kit, utilize silica membranes that bind the RNA. Samples are lysed in a buffer that contains guanidinium salts that inhibit RNase activity and the RNA is bound to the membrane by passing the lysate through the membrane using centrifugal force. Wash solutions are subsequently passed through the membrane and discarded. An appropriate elution solution is applied and the sample is collected into a tube by centrifugation. Columns are convenient, easy to use and can be automated, but have a propensity to clog and have a fixed binding capacity.
Determination of RNA yield, purity and integrity
The yield of total RNA may be determined spectrophotometrically at 260nm, whereby 1 unit of absorbance (A260) = 40µg of single stranded RNA/ml. The purity can also be determined spectrophotometrically from the ratio of the relative absorbance at 260 to 280nm and 260 to 230nm. 260/280 ratios measure the level of protein contamination in the sample, good quality RNA will have an A260/A280 ratio in the range of 1.7 to 2.1. 260/230 ratios tell you whether contaminants from the preparation are in the sample (guanidinium salts), good quality RNA will have an A260/A230 ratio above 1.5, however this ratio does not always predict success in downstream applications. One thing to note is that the A230 is often constant for purification kit used, while the amount of RNA can vary, depending on the sample source, so even if the A260/A230 ratio is below 1.5, the RNA may still be of sufficient quality for downstream applications.
RNA integrity can be assessed by running 2-4µg of a total RNA sample on an agarose denaturing gel. The RNA may be visualized by ethidium bromide staining, which reveals the ribosomal RNA bands. These bands can vary depending on the organism the RNA was extracted from. In general, for good quality RNA the bands should be distinct, with no smearing underneath them and the 28S band (larger) should be approximately twice as intense as the 18S band.
Alternatively, RNA quality and integrity can be checked using commercially available automated systems: the 2100 Bioanalyser (Agilent) or Experion (Bio-Rad). Both platforms determine RNA quality using a numerical system which represents the integrity of RNA. The Bioanalyzer offers the RIN algorithm (RNA Integrity Number) on the Bioanalyzer 2100 whereas the Experion offers an algorithm for calculating the RNA Quality Index (RQI).
Intact RNA does not guarantee good results because the RNA sample may contain inhibitors. Reverse transcription inhibitors such as SDS, EDTA, formamide and pyrophosphate can be removed by additional ethanol precipitation and washing. Inhibitors that reduce downstream reaction efficiencies, particularly of very sensitive assays such as real-time PCR or real-time RT-PCR (SensiFAST) can be determined by amplifying from a 10-fold serial dilution. The curve will be flatter (less than doubling each round of PCR) at higher template concentrations, shifting the Ct values to the right resulting in a low amplification efficiency on a standard curve. If inhibitors are detected, further purify the RNA preparation or use lower template concentration where it is known that PCR inhibition does not affect the real-time PCR results.
DNA contamination of purified RNA can also be a problem, especially when working in microbial genetics, where PCR primers cannot be designed to cross intron-exon boundaries. The level of contamination can be determined by running a PCR with the RNA directly, where the presence of an amplified product indicates genomic DNA contamination. If the amount of DNA in the RNA samples is unacceptably high, DNase treatment needs to be carried out. If this is a continuous problem (e.g. when RNA is extracted from DNA rich tissue such as spleen), then on-spin column DNase removal systems are an effective way to remove the DNA before elution of RNA.
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