Purifying Small RNAs: Using Mature MicroRNAs for Microarray Analysis
Small RNA analysis methods (e.g., hybridization assays, expression profiling) require various RNA input amounts and sizes. Small RNA fractions often include tRNA, 5S rRNA, 5.8S rRNA, small mRNA fragments, and precursor miRNAs. The presence of these RNA species does not affect Northern analysis and solution hybridization; however, they can obscure results of some experiments, particularly microarray studies. To obtain mature miRNA, use the flashPAGE™ Electrophoretic Fractionator, which purifies small RNA species (15–35 nt range) from total RNA samples. Here we show how an RNA fraction that contains only mature miRNA can provide better miRNA profiling results than an enriched small RNA (or total RNA) sample. This is just one example of the increased sensitivity gained by further removing unwanted RNA species from the sample to be analyzed. These sensitive methods may be required for detection and identification of less highly expressed miRNAs.
The correlation stays high as the amount of input is decreased from 10 to 1 µg with samples purified with the flashPAGE Fractionator (Figure 2B), but drops radically for those samples merely enriched for small RNA (Figure 2A). This drop is due to low-intensity signals as indicated by the higher correlation from the enriched samples (Figure 2A) when only those signals with very high intensities over 4500 quanta are compared.
As more microRNAs (miRNAs) are discovered, it is becoming clear that the first wave of miRNAs that were identified are expressed at a higher level than the ones now being discovered. This means that determining expression levels of many (perhaps the vast majority) of miRNAs will require a highly-sensitive method. The flashPAGE Fractionator, especially in conjunction with the mirVana miRNA Microarray System, is designed for that purpose.
Kerri Keiger, Patricia Powers, Rick Conrad • Ambion, Inc.