by Bee Na Lee, eGene, Inc.
Importance of RNA quality analysis
Determining the integrity of RNA is an essential step before gene expression analysis, because RNA quality can have a tremendous effect on downstream microarray analysis, data interpretation of gene expression profiling, and disease diagnosis. In addition to microarray analysis, real time PCR amplification analysis, reverse transcriptional experiments, and northern blot analysis rely on the high quality of the RNA samples.
The traditional method for performing RNA quality checking is slab gel agarose electrophoresis. However, preparation and manipulation of formaldehyde agarose gels can be tedious and presents exposure to biohazards. In addition, it can be difficult to measure RNA integrity by judging the intercalating intensity of ethidium bromide on the agarose gel.
Another method for determining RNA quality is the use of biochip-based electrophoresis systems. While such systems may provide high speed and sensitivity, they handle only twelve samples per chip and require manual sample preparation and loading.
In this post-genomic era, the availability of human genome information has enabled researchers to perform high throughput gene analysis for detecting diseases, analyzing mutations, screening drug targets, determining drug efficacy in clinical trials, and performing other applications.
An automated approach
The HDA-GT12 (
eGene Inc., Irvine, CA) is a multi-channel, real-time, light-induced fluorescent detection capillary electrophoresis system that provides a reliable method for routine RNA quality analysis. In addition to quality analysis, the system assures amplification efficiency and yield of cRNA and quality control of the fragmentation cRNA to ensure the validity of the chip hybridization data.
Three components make up the system — an analyzer, gel cartridge, and software. The GC-RNA
QC Gel Cartridge offers a gel matrix that enables efficient analysis of total RNA, cRNA, and fragmented RNA. The RNA analysis method has been embedded in BioCalculator software, which controls operation of the instrument and checks RNA quality including estimation of peak ratio and concentration.
An automated sample loading mechanism facilitates hands-free operation. No gel or buffer preparation is required. Twelve samples are separated in 10-minutes, and 96 samples are separated in 90 minutes. A real-time digital data collection mechanism displays both gel view and electropherogram. The system calculates the ratio of the two ribosomal bands and concentration after the analysis. It can be used for both RNA and single- and double-stranded DNA fragment analysis. As many as 96 samples can be handled in a single process.
Less than a nanoliter of sample is consumed. Based on throughput needs, one can choose four, eight, or twelve multi-capillary cartridges. The GC-RNA
QC 4 and 8 Capillary Gel Cartridge has the separation and detection capability to do 100 runs for handling up to 1,200 samples, while the GCK-RNA
QC Gel Cartridge can do 150 runs for handling 600 samples.
RNA sample preparation
Total RNA from 2.5 × 10
6 of Hela cells treated or untreated with radiation was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). Five micrograms of total RNA were used for cRNA synthesis and followed by fragmentation according to the protocol for hybridization on the HG-133 gene chip (Affymetrix, Santa Clara, CA).
One μl of total RNA (0.5μg/l-1μg/l) was denatured in the 1?μl of the loading buffer at 70 C for 2 minutes (Sigma, St. Louis, MO), and 8?μl of RNA dilution buffer (eGene) was added to bring up the total volume of 10?μl.
RNA sample analysis
The denatured total RNA samples were placed in the sample tray and injected through multiple capillary channels. BioCalculator software analyzed the samples using the total RNA analysis method.
Discussion and conclusionRNA quality analysis using HDA-GT 12 is a fully automated process after RNA samples are denatured. No sample loading or gel preparation is required. The total analysis time for twelve samples, including the sample denaturing time, is less than fifteen minutes. The 12-capillary RNA QC Gel Cartridges can be reused for up to 100 runs or for total 1,200 samples. These pre-made gel cartridges contain stable fluorescent dye gel matrix that can be stored at room temperature for up to six months.
About the author
Bee Na Lee received her PhD in microbiology from Texas A&M Univeristy and is system application manager at eGene, Inc. More information about RNA quality control and analysis is available from:eGene949-250-8686www.egeneinc.com/