Using the Cells-to-cDNA Kit, Dr. Alex Bortvin from the Whitehead Institute (Cambridge, Massachusetts) has successfully carried out RT-PCR amplification of an oocyte-specific gene and a housekeeping gene from individual mouse oocytes. Dr. Bortvin made some modifications to the Cells-to-cDNA protocol for this application. One fertilized oocyte (in 1 l of culture medium) was added to 5 l of Cell Lysis Buffer and overlaid with oil to prevent evaporation. After cell lysis, DNase I treatment was carried out with a 1:10 dilution of the enzyme at 37C for 30 min. cDNA was synthesized according to the Cells-to-cDNA protocol.
In order to ensure that clean templates for cDNA library synthesis and multiplex PCR applications was obtained, Dr. Bortvin purified the first-strand cDNA using a PCR purification kit (note that this should not normally be necessary). This step enabled him to use the PCR components and conditions that he had previously optimized for his gene of interest. PCR was carried out with gene-specific primers for 50 cycles. A "minus-RT" control reaction was included to rule out the presence of any trace genomic DNA contamination; none was seen (data not shown).
As shown in Figure 1, (top, right) using the described protocol, Dr. Bortvin was able to easily detect both his gene-specific transcript and the GAPDH housekeeping mRNA from single oocytes using the Cells-to-cDNA Kit protocol.
Figure 1. Gene-specific RT-PCR on a Single Mouse Oocyte Using the Cells-to-cDNA Kit. 10 l of each PCR was electrophoresed for 25-30 minutes in a 2% agarose gel in 1X TBE buffer containing ethidium bromide.
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