Transcriptional profiling by the two-step RT-PCR method. Total RNA from diseased or healthy tissues or from flow-sorted or laser-captured cells is isolated and reverse-transcribed with random hexamers or gene-specific primers. Then, cDNA product is PCR-amplified with 100–300 gene-specific primer sets (same as in RT reaction). Multiplex amplification is monitored carefully to evade plateau phase of the PCR. All gene-specific primers for multiplex RT-PCR are designed to have Tm = 60°C and produce small cDNA amplicons: 100–250 bp. Gene quantification via real-time PCR is performed on small aliquots of generated cDNA product with nested TaqMan primers in 96- or 384-well plate format using ABI Prizm 7700 or ABI Prizm 7900. RTF and RTR are forward and reverse primers for RT-PCR, TMF and TMR are nested TaqMan primers, and TMP is a TaqMan probe. The probe has a Fluorescein reporter dye at 5′-end (FAM) and a Black Hole Quencher at 3′-end.
