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You finally have your RNA in hand. Now what? The success of downstream applications, such as microarrays and quantitative RT-PCR, relies upon having high quality, intact RNA. So it is worth your while to make sure you have a good RNA preparation before jumping into your experiments. There are three things you want to check your RNA for: quantity, purity and integrity. And here is the Bitesize guide on how to do it…
Checking RNA quantity and purity
The traditional and least expensive way to determine RNA quantity and purity is to measure UV absorption of the sample using a spectrophotometer.
A260 for quantification: RNA has a maximum absorption at 260nm and RNA concentration is determined by the OD reading at 260nm as given by the following conversion: an A260 of 1.0 is equivalent to 40 µg/mL of RNA.
A280 for protein contamination:
In addition to the OD260, measurements should also be taken at 280nm and 230nm. The A260/A280 ratio is an indication of the level of protein contamination in the sample. Pure RNA has an A260/A280 ratio of 2.1, however values between 1.8-2.0 are considered acceptable for many protocols.
A230 for other stuff: In addition to protein contaminants, RNA preparations can also contain contaminants such as guanidine salts and phenol (used in RNA isolation protocols). A high peak at A230 indicates contamination with either of these. The ideal A260/A230 ratio is greater than 1.5.
Nanodrops, and all that: Traditional spectrophotometers require a sample of volume of at least 200?L, depending upon the cuvette used. However the NanoDrop is an excellent spectrophotometer for measuring RNA levels as it requires only 1-2 µL of sample. The NanoDrop also contains preset programs for RNA measurements that automatically read the OD at 230, 260, and 280 and calculate the concentration of the RNA and A260/A280 and A260/A230 ratios. Many departments and individual labs now contain the NanoDrop or similar instruments.
And finally, 3 words of caution:
- UV absorbance measurements can change depending upon the pH of the RNA solution. The best results are obtained when RNA is solubilized in TE buffer.
- Always blank the spectrophotometer with the same solution that the RNA is diluted in.
- And remember that RNA concentrations below 20µg/mL may not give reliable readings.
Checking RNA Integrity
Degraded RNA doesn’t perform well in downstream applications, therefore it is important to check the integrity of your RNA preparation.
Cheap and cheerful: Agarose: The least expensive method for checking RNA integrity is to run the RNA on a 1% standard agarose gel and examine the ribosomal RNA (rRNA) bands (formaldehyde gels are not required for this quick assessment). The upper ribosomal band (28S in eukaryotic cells and 23S in bacterial cells) should be about twice the intensity of the lower band (18S in eukaryotic cells and 16S in bacterial cells) and should be crisp and tight. If the rRNA bands are of equal intensity, then it suggests that some degradation has occurred. mRNA runs between the 2 ribosomal bands and might be seen as a smear. This is ok. However, smearing below the rRNA bands suggests that you have poor quality RNA. You also should not see higher molecular weight bands that might indicate that the RNA is contaminated with DNA.
Up and coming: Bioanalyzers: A second method to check the integrity of your RNA that is becoming more popular, especially with the explosion in microarray analyses, is to use a bioanalyzer, such as the Agilent Bioanalyzer. Bioanalyzers use small amounts of RNA (1-2µL) and microfluidics to determine the quantity and quality of RNA samples. The analyzer measures the sizes of the rRNA bands and determines an RNA Integrity Number (RIN) to standardize between RNA samples. Bioanalyzers are expensive, but they can often be found in core facilities and used for a fee.
If you find that your RNA is degraded, it is worth the investment to prepare new RNA prior performing experiments, making sure you take measures paranoid or otherwise to prevent RNAse contamination! For more in depth info on this topic, check out this article from Mo Bio