Abstract
The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe.
Highlights
The miRNAs are single-stranded RNA molecules of about 21~23 nucleotides in length that are both ancient and highly conserved [1,2]
We recently reported that the RNase protection assay and cyanine (Cy5)-labeled riboprobe for the analysis of a particular mRNA in the total RNA extracted from cells can be performed by using a commercial instrument, the Hitachi SV1210 microchip electrophoresis system, which is used to evaluate the integrity of total RNA [9]
As shown in Figure 1(B), the integrity of the cyanine 5 (Cy5)-miR-222 riboprobe and that of the total RNA isolated from a PC12 cell culture were confirmed by denaturing polyacrylamide gel
Summary
The miRNAs are single-stranded RNA molecules of about 21~23 nucleotides in length that are both ancient and highly conserved [1,2]. The RNase protection assay is employed for the quantitative analysis of particular RNA expression in a heterogeneous RNA sample extracted from cells, and it can identify RNA molecules of known sequence even at a low concentration in a sample [9]. This method involves hybridization of test RNAs to complementary. We recently reported that the RNase protection assay and cyanine (Cy5)-labeled riboprobe for the analysis of a particular mRNA in the total RNA extracted from cells can be performed by using a commercial instrument, the Hitachi SV1210 microchip electrophoresis system, which is used to evaluate the integrity of total RNA [9]. The potential of microchip electrophoresis for the analysis of miRNA expression was shown, offering highly sensitive detection and ease of operation in a short time
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