Isolation of intact RNA is essential for many techniques used in gene expression analysis. Northern analysis, cDNA library construction and cDNA labeling for microarray analysis (especially when priming with oligo(dT)) require RNA of extremely high integrity. RT-PCR and ribonuclease protection assays both involve analysis of smaller regions of RNA (generally less than 1 kb), and, therefore, are more tolerant of partially degraded RNA. Regardless of the downstream application, it is a good idea to check RNA integrity before gene expression analysis.
The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). While native (non-denaturing) gels can be used, the results can be difficult to interpret. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Nondenaturing conditions also result in bands that are not as sharp, and even multiple bands representing different structures of a single RNA species.
Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band (Figure 1, lane 3). This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a very low molecular weight smear (Figure 1, lane 2). Inclusion of RNA size markers on the gel will allow the size of any bands or smears to be determined and will also serve as a good control to ensure the gel was run properly (Figure 1, lane 1). Note: Poly(A) selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).
Figure 1. Intact vs. Degraded RNA.
Two µg of degraded total RNA and intact total RNA were run beside Ambion's RNA Millennium Markers™ on a 1.5% denaturing agarose gel. The 18S and 28S ribosomal RNA bands are clearly visible in the intact RNA sample. The degraded RNA appears as a lower molecular weight smear.
A drawback to using denaturing agarose gels to assess integrity RNA is the amount of RNA required for visualization. Generally, at least 200 ng of RNA must be loaded onto a denaturing agarose gel in order to be visualized with EtBr. Some RNA preparations, such as those from needle biopsies or from laser capture microdissected samples, result in very low yields. In these cases, it may be impossible to spare 200 ng of RNA to assess integrity before proceeding with the expression profiling application. Alternative nucleic acid stains, such as SYBR® Gold and SYBRï Green II RNA gel stain from Molecular Probes (Eugene, OR), offer a significant increase in sensitivity compared to the traditional EtBr stain in agarose gels. Using a 300 nm transilluminator (6 x 15-watt bulbs) and a special filter, as little as 1 ng of RNA can be detected with SYBR Gold and 2 ng with SYBRï Green II RNA gel stain, allowing less sample to be used for pre-experimental integrity assessment.
Figure 2. Agilent 2100 Bioanalyzer Data.
Electropherogram of a high quality, eukaryotic, total RNA sample. The 18S and 28S peaks are clearly visible at 39 and 46 seconds, respectively. The microchannels of the Bioanalyzer are filled with a sieving polymer and fluorescence dye. Samples are detected by their fluorescence and translated into electropherograms or into gel-like images (data not shown).
Alternative to Agarose Gel Analysis
Currently there exists an alternative to traditional gel-based analysis that integrates the quantitation of RNA samples with quality assessment in one quick and simple assay. The Agilent 2100 Bioanalyzer (Agilent Technologies) is the first commercially available microfluidics instrument to provide detailed information about the condition of RNA samples. Used in coordination with the RNA 6000 LabChip® (a registered trademark of Caliper Technologies Corporation), as little as 1µl of 10 ng/µl is required per analysis. In addition to assessing RNA integrity, this automated system also provides a good estimate of RNA concentration and purity (i.e. rRNA contamination in mRNA preparations) in a sample. Previously, part of an RNA sample would be used for concentration and purity measurement (by A260 spectrophotometry) and another part of the sample would be used for integrity assessment. Using the LabChip® system however, concentration, integrity and purity are analyzed simultaneously in a single 5 ng sample. Data can be displayed as a gel-like image, an electropherogram (Figure 2), and in tabular format.