Is Your RNA Intact? Methods to Check RNA Integrity
|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.|
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.
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).