Current methods for the determination of RNA quantity/quality/integrity are labor-intensive, costly, and often cannot deliver adequate information or analyze high numbers of samples. New developments in capillary electrophoresis (CE) now provide for automated RNA quality/quantity analysis in a high-throughput environment.
Spectrophotometry is the most common method for RNA quantification, where the absorbance at 260 nm gives quantity, while the ratio of 260 nm/280 nm gives a measure of purity with respect to protein or solvent contaminants. However, the absorbance method does not give any differentiating information related to DNA contamination or the degradation state of the RNA (RNA integrity). Also, fluorometric methods are commonly used to quantify RNA, but lack information on RNA integrity and often miss the presence of contaminants such as genomic DNA (gDNA).
Partially denaturing agarose gel electrophoresis is used for determining the quality of an RNA sample, wherein the ratio or visual presence of the large and small subunit (28S/18S) of the ribosomal RNA is correlated with the integrity of the RNA. However, this method is labor-intensive, requires large amounts of extracted RNA, lacks resolution, and is prone to errors in the interpretation of RNA quality.
Recent improvements to the agarose gel method include the use of microgels sandwiched within laminated tape-like cartridges, but the method still lacks resolution.
Until recently, the benchmark standard for determining RNA integrity has been based on microchannel, chip-based electrophoresis systems that provide faster run times and improved data quality compared to agarose gel electrophoresis, but require hands-on processing for priming and loading of gel, markers, and samples onto the system.
The samples are analyzed sequentially in the same separation channel, without flushing or priming between injections, which can result in sample carryover or trapped particulates and air bubbles that adversely affect subsequent separations.
Capillary electrophoresis offers advantages over both agarose and microchip electrophoresis in that gel filling and sample loading are automated. However, many commercial CE instruments are prohibitively expensive and focus on single-stranded DNA rather than RNA or double-stranded DNA analysis. Other CE instruments lack the sensitivity, dynamic range, and separation quality required for adequate RNA quality/quantity analysis.
Although all techniques can be interfaced to a robotic system, only the most sophisticated core labs can afford both the cost and expertise required to operate such systems.
Advanced Analytical Technologies recently introduced the Fragment Analyzer™ CE System. With the capability of running 12 or 96 samples per run, the Fragment Analyzer bridges the gap between a low-volume stand-alone instrument and one that is interfaced with a robotic system. The instrument features: