Please refer to the sections “Q-PCR supplies, reagents and stock solutions” and “Preparation of cDNA Standards” in the Supplementary File 1 for the detailed protocols and worksheets for this section.
Total RNA is prepared from cells or frozen tissues with RNA STAT-60 according to the manufacturer′s instructions. For a typical assay, 4µg of total RNA is treated with a 1:5 dilution of RNase-free DNase I in the presence of 4.2mM MgCl2 in a final volume of 20µl. This is performed in 0.2ml thin-walled PCR tubes in a standard thermocycler at 37°C, 30 min., 75°C, 10 min., and 4°C hold. The reverse-transcription mix consisting of 1X First Strand Buffer, 10mM DTT, 200U of SuperScript RTII reverse transcriptase, 2mM dNTPs and 0.08µg/µl random hexamers is then added directly to the tubes with the DNAse-treated RNA for a final volume of 100µl. The cDNA synthesis is carried-out in the thermocycler at 25°C for 10 min., 42°C for 50 min., 72°C for 10 min., and 4°C hold. Following the reverse-transcription, DEPC-treated H2O is added to the unknown samples to bring the volume to 200µl, and the cDNA concentration to 20ng/µl. (Note that samples used as cDNA standards are not diluted prior to making the 5-fold dilution series used in primer validation and standard curve assays.) This will result in enough template cDNA to test approximately 40 QPCR targets.
Total RNA is prepared from cells or frozen tissues with RNA STAT-60 according to the manufacturer′s instructions. For a typical assay, 4µg of total RNA is treated with a 1:5 dilution of RNase-free DNase I in the presence of 4.2mM MgCl2 in a final volume of 20µl. This is performed in 0.2ml thin-walled PCR tubes in a standard thermocycler at 37°C, 30 min., 75°C, 10 min., and 4°C hold. The reverse-transcription mix consisting of 1X First Strand Buffer, 10mM DTT, 200U of SuperScript RTII reverse transcriptase, 2mM dNTPs and 0.08µg/µl random hexamers is then added directly to the tubes with the DNAse-treated RNA for a final volume of 100µl. The cDNA synthesis is carried-out in the thermocycler at 25°C for 10 min., 42°C for 50 min., 72°C for 10 min., and 4°C hold. Following the reverse-transcription, DEPC-treated H2O is added to the unknown samples to bring the volume to 200µl, and the cDNA concentration to 20ng/µl. (Note that samples used as cDNA standards are not diluted prior to making the 5-fold dilution series used in primer validation and standard curve assays.) This will result in enough template cDNA to test approximately 40 QPCR targets.
