Quantitate and Profile miRNA without RNA Isolation
The lysates are then ready for reverse transcription or storage at –20ºC for up to 5 months. Because the samples are not subjected to an RNA purification process, all RNA species, including the small RNA fraction, remain present for analysis. Additionally, the samples can be processed directly in culture plates (96 or 384 wells), which reduces sample handling and the risk of sample loss or transfer errors. No heating, washing, or centrifugation is required.
Additionally, the ability to distinguish between highly homologous mature miRNA targets is essential for accurate miRNA expression analysis. The TaqMan MicroRNA Cells-to-CT Kit, along with TaqMan MicroRNA Assays, delivers the specificity required to make this distinction (Figure 1).
Figure 1. Single-base Discrimination of TaqMan® MicroRNA Assays with the TaqMan MicroRNA Cells-to-CT™ Kit. The ability of TaqMan MicroRNA Assays to distinguish between the highly homologous let-7 family of miRNAs was not affected by the use of TaqMan MicroRNA Cells-to-CT Kit lysates. No difference was observed in the relative detection levels of synthetic let-7 miRNAs in the presence of TaqMan MicroRNA Cells-to-CT Kit lysates (A) or purified miRNA (B) generated from 1000 HeLa cells.
Results Equivalent to Purified RNA
Figure 2. Detection of MicroRNA by RT-PCR is Equivalent in TaqMan® MicroRNA Cells-to-CT™ Kit Lysates and Purified RNA. TaqMan MicroRNA Assays were used to compare the detection of miRNA in TaqMan MicroRNA Cells-to-CT Kit lysates (y axis) and purified RNA (x axis) generated from 1000 HeLa Cells. Each data point represents the CT values from biological duplicates and technical duplicates (n=4) for each of the 111 TaqMan MicroRNA Assays tested. R2=0.931, Slope=0.977.
Broad Dynamic Range
Figure 3. Linear Real-Time RT-PCR Using 10–100,000 Cells With the TaqMan® MicroRNA Cells-to-CT™ Kit. A dilution series of 10–100,000 HeLa cells was processed in duplicate with the TaqMan MicroRNA Cells-to-CT Kit, and by traditional RNA purification. Two representative assays [A: Assay miR-16 (P/N 4373121), and B: Assay RNU48 (P/N 4373383)] were performed in duplicate using each RNA sample source, and averaged values were plotted as a function of cellular input.
Laura Chapman, Annalee Nguyen, and Richard Fekete • Applied Biosystems, Austin, TX