cDNA from blood RNA was initially probed for presence of human DOP receptor transcripts using TaqMan probes, Hs00538331_m1 (Thermo Fisher Scientific, UK), which revealed amplification of product, i.e. presence of the human DOP receptor. However this was a false positive with amplification measured in parallel non-template control samples, in which the reverse transcriptase enzyme had been removed in the conversion of whole blood RNA to cDNA, suggesting the amplification in both samples to be genomic DNA. The same false positive amplification was seen with further commercially available TaqMan probes for the DOP receptor, Hs00357182_mh (Thermo Fisher Scientific, UK) and Hs_OPRD1_QF_1 (Qiagen, UK). Such gDNA amplification was despite RNA samples being processed with a DNase enzyme, designed to remove DNA contaminates, provides a cautionary tale and the need for using the correct negative controls. To distinguish cDNA from gDNA primers were designed that span an exon/exon boundary on the Homo sapiens opioid receptor, delta 1 (OPRD1) mRNA sequence. While these primers initially yielded amplification in some NTC samples, through a series of validating experiments the stringency with which they amplified specifically cDNA for the DOP receptor was increased. Using the STD-DOP-1 primer pair, under the correct experimental conditions, no amplification of the DOP receptor transcript could be detected, whilst a robust and consistent amplification was determined in positive control samples, cDNA from CHOhDOP cells. Again this highlights the importance and possibility of detecting a false positive if the correct experimental conditions are not defined. Recent studies have indicated that the nociceptin system has a role in inflammation and sepsis. 21 Clinical evidence from ICU patients diagnosed with sepsis has shown how there is a decrease in mRNA encoding for the NOP receptor in septic patients when compared with a matched control volunteer. Previously we have demonstrated that while PBMCs lack expression of classical opioid receptors (MOP, DOP, KOP), they do express NOP receptor
cDNA from blood RNA was initially probed for presence of human DOP receptor transcripts using TaqMan probes, Hs00538331_m1 (Thermo Fisher Scientific, UK), which revealed amplification of product, i.e. presence of the human DOP receptor. However this was a false positive with amplification measured in parallel non-template control samples, in which the reverse transcriptase enzyme had been removed in the conversion of whole blood RNA to cDNA, suggesting the amplification in both samples to be genomic DNA. The same false positive amplification was seen with further commercially available TaqMan probes for the DOP receptor, Hs00357182_mh (Thermo Fisher Scientific, UK) and Hs_OPRD1_QF_1 (Qiagen, UK). Such gDNA amplification was despite RNA samples being processed with a DNase enzyme, designed to remove DNA contaminates, provides a cautionary tale and the need for using the correct negative controls. To distinguish cDNA from gDNA primers were designed that span an exon/exon boundary on the Homo sapiens opioid receptor, delta 1 (OPRD1) mRNA sequence. While these primers initially yielded amplification in some NTC samples, through a series of validating experiments the stringency with which they amplified specifically cDNA for the DOP receptor was increased. Using the STD-DOP-1 primer pair, under the correct experimental conditions, no amplification of the DOP receptor transcript could be detected, whilst a robust and consistent amplification was determined in positive control samples, cDNA from CHOhDOP cells. Again this highlights the importance and possibility of detecting a false positive if the correct experimental conditions are not defined. Recent studies have indicated that the nociceptin system has a role in inflammation and sepsis. 21 Clinical evidence from ICU patients diagnosed with sepsis has shown how there is a decrease in mRNA encoding for the NOP receptor in septic patients when compared with a matched control volunteer. Previously we have demonstrated that while PBMCs lack expression of classical opioid receptors (MOP, DOP, KOP), they do express NOP receptor