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Quantitate and Profile miRNA without RNA Isolation
- 10-minute sample preparation; no RNA isolation
- Results equivalent to purified RNA
- Linear detection from 10–100,000 cells
- Designed for use with TaqMan® MicroRNA Assays
MicroRNA (miRNA) expression profiling directly from cultured cells without RNA purification is quick, easy, and sensitive with the TaqMan® MicroRNA Cells-to-CT™ Kit. This is the first product to offer a simple, complete workflow for miRNA expression analysis for manual or automated applications.
The kit includes components for cell lysis, which remove genomic DNA and preserve RNA integrity, and for real-time PCR analysis [e.g., TaqMan MicroRNA Reverse Transcription (RT) Reagents and TaqMan Universal PCR Master Mix]. TaqMan MicroRNA Assays targeting specific miRNAs must be purchased separately. The TaqMan MicroRNA Cells-to-CT Kit was extensively tested and validated with TaqMan MicroRNA Assays.
Simple Protocol
The protocol uses a simple 10-minute sample preparation procedure, regardless of whether the experiment involves 10 or 100,000 cultured cells/sample. 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 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.
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.
Superior Specificity
TaqMan MicroRNA Assays are designed with a target-specific, stem-loop reverse-transcription primer that addresses challenges associated with the short length of mature miRNAs. This stem-loop structure is specific to the targeted mature miRNA, which distinguishes it from methods that indiscriminately extend and prime all RNAs, including biologically inactive precursor miRNA, in a sample.
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.
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
This new kit has been extensively tested with a broad selection of TaqMan MicroRNA Assays. Results from 111 assays demonstrate similar expression patterns and equal sensitivity between both TaqMan MicroRNA Cells-to-CT Kit lysates and purified RNA (Figure 2). This workflow enables unsurpassed miRNA expression evaluation with ~700 mammalian TaqMan MicroRNA Assays currently available.
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. R 2=0.931, Slope=0.977.
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. R 2=0.931, Slope=0.977.
Broad Dynamic Range
Because miRNA expression levels can vary widely, depending upon sample type, developmental stage, or disease progression, sensitive tools for miRNA expression analysis are critical. When matched with TaqMan MicroRNA Assays, the TaqMan MicroRNA Cells-to-CT Kit provides unrivaled flexibility, enabling miRNA quantitation with as few as 10 or as many as 100,000 input cells. To achieve maximum assay sensitivity, sample lysate can comprise up to 33% of a singleplex RT reaction volume or 20% of a multiplex RT reaction volume. The linearity and dynamic range of detection from kit lysates across 5 logs of cellular input per lysis reaction can be seen in Figure 3, with results equivalent to purified RNA. In summary, the broad dynamic range of TaqMan MicroRNA Assays is not compromised by use of TaqMan MicroRNA Cells-to-CT Kit lysates in place of purified RNA.
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.
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.
Seamless Workflow
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 reagents for sample preparation, reverse transcription, and real-time PCR.
Scientific Contributors
Laura Chapman, Annalee Nguyen, and Richard Fekete • Applied Biosystems, Austin, TX
Scientific Contributors
Laura Chapman, Annalee Nguyen, and Richard Fekete • Applied Biosystems, Austin, TX