Traditionally, RNA integrity has been evaluated using formaldehyde agarose-based gel electrophoresis of the total RNA sample. However, such traditional techniques require large amounts of sample, lack sensitivity, take up a significant amount of time, and require a trained eye and expertise for judging RNA integrity. Quantitation of RNA has typically been done using spectrophotometric methods. Microfluidic platforms, such as the Experion system, have eliminated the need to use two separate techniques to evaluate integrity and concentration of RNA. A microfluidic system enables use of much smaller amounts of sample, leaving remaining sample for downstream applications, and it enables quick assessment of concentration, integrity, and visual documentation in all samples. Moreover, microfluidic platforms enable sensitivity down in the pg range and reduce hands-on time and data imaging and analysis. Two RNA analysis kits are available. The sensitivity of the Experion StdSens RNA analysis kit is in the ng range, while the pg range sensitivity of the Experion HighSens RNA analysis kit is more appropriate for samples that are very precious and at low concentrations. In addition to total RNA, the Experion RNA kits can also be used to evaluate mRNA (using mRNA assays) and will generate information such as mRNA concentration, rRNA % contamination, and size distribution and integrity (peak profile).
In addition to RNA, dsDNA can also be analyzed on the Experion system. The system makes analysis of DNA amplicons from PCR reactions and other DNA-related applications such as RFLP less time-consuming and hands-on. There are two DNA analysis kits — one that is more appropriate for dsDNA fragments between 15 and 1,500 bp (Experion DNA 1K analysis kit) and another that is more appropriate for fragments between 50 and 17,000 bp (Experion DNA 12K analysis kit) — and both have sensitivities in the ng range.
In 2009, leading researchers in the field of quantitative real-time PCR emphasized the importance of improving the reproducibility and quality of publications and work. Recognition of the need for higher consistency and standardization of qPCR experiments led to the development of MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines (Bustin et al. 2009).
In line with methods that aid in the consistency and reproducibility of data, and the quality control of starting materials, the Experion RNA assays enable electrophoretic separation, visualization, quantitation, and objective judgement of total RNA integrity via RQI number.
After each eukaryotic total RNA run, the Experion system automatically generates an RQI number based on the profile of each sample. RQI is a number ranging from 1 to 10, with 1 being the most degraded RNA profile and 10 being the most intact, highest-integrity RNA profile. The cutoff RQI number to determine whether or not a sample can be used for downstream applications must be empirically identified. Once the acceptable cutoff is identified, RNA with RQI numbers less than the cutoff can be eliminated. Cutoff RQI number can be different depending on the stringency and requirements of the downstream application. For example, an RQI cutoff value for total RNA to be used for qPCR may be 5, while the RQI cutoff for RNA to be used for microarray experiments may be 7. The RQI cutoff number may also be dependent on the type of sample. For example, the range for acceptable RQI values for RNA extracted from biopsy tissues may be lower than that of RNA extracted from cell cultures, simply because biopsy tissues may have more degradation from handling and harvesting conditions. See bulletins 5859 and 5452.
Using a method to assess RNA integrity before downstream applications ensures more consistent and reliable results. For example, in evaluating the effects of gene silencing via siRNA-induced suppression, using intact RNA for both the controls and sample is important and can mean the difference between accurate or inaccurate estimation of the silencing effect.
Impact of siRNA-targeted RNA sample quality on RT-qPCR. Experion electropherograms of intact or partially degraded total RNA from transfections with scrambled control siRNA and GAPDH-targeted siRNA are shown. Panels A–D show RT-qPCR traces obtained using the combination of scrambled control (red) and GAPDH-targeted (blue) RNA sample indicated in the two electropherograms positioned above and beside the trace. Also shown are the average measured CT values for each sample and the overall level of gene silencing, which is represented by the ΔCT value.
DNA fragments from either PCR reactions or restriction digestions (such as SNP genotyping done using RFLP) may also be separated, sized, quantitated, and analyzed using the Experion system. The Experion DNA assays can be used to analyze relative ratios of the fragments. Below, RFLP analysis of the human ALDH2 (aldehyde dehyrdrogenase type 2) gene on the Experion system shows the three types of SNPs — G/G homozygote, G/A heterozygote, and A/A homozygote — following PCR and digestion with a restriction enzyme.
ALDH2 SNP analysis using the Experion system. A, electrophoretic profile of a MboII digested PCR fragment containing codon 487 of ALDH2. — G/G homozygous DNA, — G/A heterozygous DNA, and — A/A homozygous DNA; B, G/A heterozygous DNA from different samples.
An interesting application for the Experion DNA assay was presented by Spaniolas et al. (2008), who illustrated that an automated electrophoresis system, using the DNA assay to separate PCR amplicons from various food oils, can be utilized to authenticate olive oil, enabling fast identification of the most-likely-used oil adulterants in olive oil.
Bustin SA et al. (2009). The MIQE guidelines: minimum information for publication for quantitative real-time PCR experiements. Clin Chem 55, 611–622.
Spaniolas S et al. (2008). Exploitation of the chloroplast trnL (UAA) intron polymorphisms for the authentication of plant oils by means of a lab-on-a-chip capillary electrophoresis system. J Agric Food Chem 56, 6886–6891.