- No RNA purification required
- From cells in culture to cDNA in less than 2 hours
- Detect rare messages in as few as 1 cell
- Ideal for labs not equipped for RNA isolation
The Cells-to-cDNA™ II Kit produces cDNA from cultured mammalian cells in less than 2 hours. No RNA isolation is required. This kit is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation.
Ideal for RT-PCR Applications
Quantitative RT-PCR is the most common method used for measuring mRNA levels from small numbers of cells. This type of analysis can be difficult when using RNA from these very small samples. Ambion's Cells-to-cDNA II Kit (patent pending) overcomes this limitation by integrating RNase inactivation and DNase I treatment into an RT-PCR compatible cell lysis buffer, eliminating RNA isolation altogether. Most cell lysis buffers contain strong denaturants that if carried over into enzymatic reactions would inhibit or inactivate most enzymes. If a lysis buffer without strong denaturants is used, endogenous RNase can quickly degrade cellular RNA. Ambion's Cells-to-cDNA II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly
added to a cDNA synthesis reaction. Cells-to-cDNA II is compatible with both one-step and two-step RT-PCR protocols.
Quantitative — Linear Results and No Detectable DNA Contamination
To illustrate the quantitative, linear response of Cells-to-cDNA II to variations in input cell number, a real-time quantitative RT-PCR experiment was
performed using the ABI 7700 Sequence Detection System (see figure at left). HeLa cells were grown to ~75% confluency in T-flasks and detached
by incubation with trypsin. Cells were counted, washed once with phosphate buffered saline (PBS), and serially diluted in PBS. An aliquot (5 µl) of each dilution was added to 95 µl of Cell Lysis Buffer so that the final cell concentrations ranged from 1 to 10,000 cells/µl. The samples were lysed and DNase I treated according to the standard Cells-to-cDNA II protocol. For cDNA synthesis, 5 µl of each lysate were reverse transcribed in a 20 µl reaction. GAPDH TaqMan® qRT-PCR analysis was performed on 5 µl of each cDNA preparation using SuperTaq™ DNA Polymerase in the ABI 7700
Sequence Detection System. Results of these experiments are shown in the figure at left. Plotting cycle threshold (Ct) versus cell concentration for GAPDH
yielded a standard curve with a correlation of 0.99. Thus, Cells-to-cDNA II yields linear results for 1 to 10,000 cells/µl in a Real-time qRT-PCR two
step assay. It should also be noted that the minus-template PCR control for the GAPDH experiment was negative, indicating complete removal of
genomic DNA.
Simple Procedure
The Cells-to-cDNA Kits come with everything you need to go from cultured cells to PCR-ready cDNA is less than 2 hours; no RNA isolation is required. As illustrated in figure 2, cells are first washed with PBS and then resuspended in the Cell Lysis Buffer. A brief heating simultaneously lyses
the cells and inactivates RNases. An optional DNase digestion is then performed to degrade genomic DNA, followed by a heat inactivation step. A fraction of the cell lysate is then used in a reverse transcription reaction with the included reagents.
A Complete Kit for Cell Lysis and cDNA Synthesis
The Cells-to-cDNA II Kit contains all the necessary reagents for 40 or 100 reverse transcription reactions, including M-MLV RT and oligo dT18 and random decamer primers. The kit also contains an Armored RNA® control and complementary primers. This control can be added to the cells to monitor inactivation of endogenous and exogenous ribonucleases and, along with the primers, can be used to examine reverse transcription efficiency. From 1 to 50,000 cells can typically be used in each Cells-to-cDNA reaction.
SuperTaq and SuperTaq Plus are trademarks of and are manufactured by Enzyme Technologies Ltd. and sold under licensing arrangements with F. Hoffman-La Roche, Ltd., Roche Molecular Systems, Inc. and the Perkin-Elmer Corporation. Ambion, Inc. is a distributor of Enzyme Technologies, Ltd., except in the following countries: Austria, Benelux, Denmark, Greece, Sweden, Switzerland, Taiwan, and the United Kingdom.
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