Gelgreen dna dye

The expression of the nuclear progesterone receptor (nPGR) was investigated by reverse transcription PCR (RT-PCR). Two previously described RT-PCR assays were used to ensure the detection of all known PGR transcript isoforms A–D (accession numbers NM_000926.4, NM_001202474.3, NM_001271161.2, and NM_001271162.1). The first assay named “PGR,” by Latil [35 (link)], detects the isoforms A–C, whereas the second RT-PCR “PR-A + B” [36 (link)] amplifies A, B, and D (Supplemental Table 1). The reaction mixes for RT-PCR were identical to those of RT-qPCR, except that the probe and ROX reference dye were omitted and replaced with water. The PCR temperature profiles were performed as described in the original papers [35 (link), 36 (link)]. PCR products were separated on a 2% agarose gel, stained with the GelGreen DNA dye (Biotium, Hayward, USA), and visualized under blue light excitation. RNA from T-47D cells was included as a positive control for the PGR RT-PCR. The RPL27 gene was used as internal standard to measure the RNA input quantity.