RNA Integrity Assessment via Agarose Gel Electrophoresis
Updated 3/15/2024 01:19 PM
Introduction
Materials
- RNA samples
- TAE buffer
- Agarose
- Ethidium Bromide (EtBr) (10mg/mL)
- DNA loading buffer (10X)
- 1.9mM xylene cyanol, 1.5mM bromophenol blue, 25% glycerol in sterile dH2O
- DNA ladder (1kb)
Procedure
Agarose Gel preparation
- Add 1.0% w/v agarose to 1x TAE buffer (e.g. 0.5g in 50mL)
- incubate at room temperature for 5 min, with occasional swirling
- Heat the suspension to melt the agarose
- Allow solution to cool before adding ethidium bromide to a final concentration of about 0.5µg/mL
- Pour solution into gel mold and allow the ‘agarose gel’ to solidify
Load and run RNA samples
- Load RNA sample mixed with 10x DNA loading buffer to a final concentration of 1x.
- Load DNA ladder
- Electrophorese gel in 1x TAE buffer at 100V for approximately 35 min.
- Visualize bands by UV transillumination
Determine if 28S & 18S bands are present. Use gel image analyzer to determine ratio if needed.
High-quality RNA isolation is crucial for gene expression analysis techniques like Northern analysis, cDNA library construction, and cDNA labeling for microarray analysis. These methods demand RNA with high integrity. While RT-PCR and ribonuclease protection assays can tolerate partially degraded RNA, it's advisable to check RNA integrity for any gene expression analysis.
To assess RNA quality, running intact total RNA on a denaturing gel should produce clear 28S and 18S rRNA bands with a 2:1 ratio (28S:18S), indicating intact RNA. Partially degraded RNA will appear smeared, lacking sharp rRNA bands or the 2:1 ratio, while completely degraded RNA appears as a low molecular weight smear. Including RNA size markers on the gel helps determine band sizes and ensures proper gel running.
In eukaryotic RNA, three distinct bands on gel electrophoresis signify high-quality RNA: 28S rRNA (approximately 4.8kb), 18S rRNA (around 2.0kb), and 5.8S/5S RNA. Transfer RNAs may or may not be visible. Smeared bands or a 28S:18S ratio below 2:1 indicate poor RNA quality.
To evaluate the quality of RNA without the use of toxic chemicals, samples are run in a Agarose Gel and product segregation is used to estimate RNA integrity. Be aware that in a non-denaturing gel, the RNA will not segregate strictly on particle size due to secondary structures of the molecules. This method is for an estimation of quality only and the location of the banding in relation to a base pair ladder does not allow confident determination of the size of the RNA fragment. If true fragment size determinations are required, RNA quality should be evaluated on a denaturing gel electrophoresis system.
Note: Poly(A) selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6kb to 0.5kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2kb being the most intense (this smear is sometimes apparent in total RNA samples as well).
References: TBA