The Ambion® TaqMan® MicroRNA Cells-to-CT™ Kit makes it possible to perform microRNA (miRNA) expression profiling directly from cultured cells without RNA purification. The kit is the first to offer a simple, complete workflow for miRNA expression analysis from a few samples, or it can easily be incorporated into automated, high throughput applications.
• Complete, validated solution—Optimized workflow includes cell lysis reagents, DNase, TaqMan® MicroRNA RT reagents, and TaqMan® Universal PCR Master Mix
• Fast, simple, and convenient—Prepare samples in 10 minutes; eliminate tedious RNA isolation
• Gold standard specificity—Achieve superior results with TaqMan® MicroRNA reagents
• Robust performance—Results equivalent to purified RNA
• Broad input dynamic range—Linear detection from 10 to 100,000 cells
Complete, Validated Solution
Featuring a unique method for lysing cultured cells while removing genomic DNA and preserving RNA integrity, the TaqMan® MicroRNA Cells-to-CT™ Kit contains TaqMan® MicroRNA reverse transcription (RT) reagents and TaqMan® Universal PCR Master Mix for real-time PCR analysis. The TaqMan® MicroRNA Cells-to-CT™ Kit has been extensively tested and validated with TaqMan® MicroRNA Assays, sold separately.
Simple, Convenient 10-Minute Sample Preparation
The TaqMan® MicroRNA Cells-to-CT™ Kit incorporates a simple 10-minute sample preparation procedure (Figure 1) for use with 10–100,000 cultured cells⁄sample, either in plates or tubes. Cells are washed in PBS and lysed for 8 minutes at room temperature; DNase treatment can be performed concurrently. Lysis is terminated at room temperature by a 2-minute incubation with Stop Solution.
The TaqMan® MicroRNA Cells-to-CT™ Kit lysates are now ready for reverse transcription or can be stored at -20°C for up to 5 months. Since samples are not subjected to an RNA purification process, all of the small RNA species are available for analysis. Because samples can be processed directly in culture plates (96 or 384 wells), sample handling is reduced, and the risk of sample loss or transfer error is minimized. No heating, washing, or centrifugation is required; the kit greatly simplifies a traditionally time-consuming, labor intensive process and reduces it to 10 minutes.
Gold Standard Specificity—Superior Results with TaqMan® MicroRNA Reagents
Applied Biosystems has developed a state-of-the-art solution to the fundamental problem associated with the short length of mature miRNAs. TaqMan® MicroRNA Assays (sold separately) include a unique, target-specific stem-loop reverse-transcription primer (Figure 2). The stem-loop structure is designed to incorporate specificity for the targeted mature miRNA, in contrast to other methods that indiscriminately extend and prime all RNA in a sample. The TaqMan® MicroRNA RT reagents, included in the TaqMan® MicroRNA Cells-to-CT™ Kit, were specifically designed to work in conjunction with this innovative assay design.
The ability to distinguish between highly homologous mature miRNA targets is required for accurate miRNA expression analysis. The TaqMan® MicroRNA Cells-to-CT™ Kit, in conjunction with the TaqMan® MicroRNA Assays, delivers the specificity needed to distinguish between highly homologous targets, as seen in Figure 3. In this example, lysates from the TaqMan® MicroRNA Cells-to-CT™ Kit or purified miRNA were used in real-time PCR using TaqMan® MicroRNA Assays to detect single base differences on exogenously added miRNA templates. Specificity obtained with TaqMan® MicroRNA Cells-to-CT™ Kit lysates (Figure 3A) was equivalent to that using purified RNA (Figure 3B).
Robust Performance—Results Equivalent to Purified RNA
This new kit has been extensively tested with a broad selection of TaqMan® MicroRNA Assays and demonstrates performance equivalent to that obtained with purified RNA (Figure 4). The TaqMan® MicroRNA Cells-to-CT™ Kit workflow enables unsurpassed miRNA expression evaluation with > 900 available TaqMan® MicroRNA Assays.
Broad Input Dynamic Range—Linear Detection from 10 to 100,000 Cells
Because miRNA expression levels can vary widely due to sample type, developmental stage, or disease progression, it is important to have sensitive tools for miRNA expression analysis. Now, miRNA targets that vary from a few copies to millions of copies can be accurately quantitated in the same experiment.
The TaqMan® MicroRNA Cells-to-CT™ Kit works robustly with as few as 10 cells, and up to 100,000 cells. Furthermore, to maximize sensitivity, the TaqMan® MicroRNA Cells-to-CT™ Kit allows up to 33% of the RT reaction volume to be comprised of sample lysate (singleplex RT). Figure 5 demonstrates the linearity and sensitivity of detection from TaqMan® MicroRNA Cells-to-CT™ Kit lysates across 5 logs of cellular input per lysis reaction; results were equivalent to purified RNA. Additionally, the broad dynamic range of TaqMan® MicroRNA Assays is not compromised by TaqMan® MicroRNA Cells-to-CT™ Kit lysates (data not shown).
Proven Performance, Proven Together
All components of the TaqMan® MicroRNA Cells-to-CT™ Kit have been optimized for consistent and reliable performance. This removes the guesswork involved in assembling separate kits for sample preparation, reverse transcription, and real-time PCR. The TaqMan® MicroRNA Cells-to-CT™ Kit has been validated with TaqMan® MicroRNA Assays for added quality assurance.