Heparinase treatment of RNA before quantitative real-time RT-PCR.

Vol. 35, No. 6 (2003) BioTechniques 1141 the ABI PRISM® 7000 and the TaqMan Universal PCR Master Mix protocols (Applied Biosystems). After discovering that the RNA from the thecal cells had vascular endothelial growth factor (VEGF) amplification and that RNA from granulosa cells had no VEGF amplification, the 18S rRNA amplification (TaqMan Pre-Developed Assay Reagents; Applied Biosystems), which is used as a positive control for eukaryotic gene expression and to normalize RNA concentrations, was performed on the granulosa samples to see if there was a problem with the RT reaction. The assay revealed that inhibition of the PCRs for the 18S rRNA assay was also present in granulosa cell RNA. An attempt to remove the unknown inhibitor(s) from the RNA using an RNA column purification clean-up procedure from Zymo Research (Orange, CA, USA) was unsuccessful. Progressive dilutions of the RT of a thecal sample that amplified well during PCR with an RT from a sample of granulosa cell RNA that was PCRinhibited resulted in the progressive inhibition of amplification of the thecal sample (Figure 2). To identify heparin as the inhibitor present in the RNA, heparin and/or polyacrylamide carrier Heparinase treatment of RNA before quantitative real-time RT-PCR