Primers and probe were tested for selectivity, specificity and sensitivity of detection of the target organism using a fluorogenic nuclease assay and a sequence detector.
One scaffold was synthesized and inhibitory binding tested using a fluorogenic ACE assay.
Media samples were analysed for total MMP activity using a fluorogenic substrate assay.
All PCR amplifications were carried out in duplicate on an ABI Prism® 7300 Sequence Detection System, using a fluorogenic 5' nuclease assay (TaqMan® probes).
Proteasome function was measured in cell lysates from ECV304 cells incubated with different doses of verapamil, doxorubicin, daunorubicin, idarubicin, epirubicin, topotecan, mitomycin C, and gemcitabine using a fluorogenic peptide assay.
Real-time PCR amplification using a fluorogenic 5' exonuclease assay was performed in triplicate on negative controls and DNA plasmid dilutions of 10 102 copies to give a total of 24 reactions per PCR experiment.
Both inhibitors blocked intracellular cathepsin activity as measured using a fluorogenic cathepsin B/L assay (Fig. 4E).
Expression levels of Fas mRNA were analyzed by quantitative real-time PCR using a fluorogenic 5'-nuclease assay (TaqMan®; Applied Biosystems, Weiterstadt, Germany) on an ABI Prism 7900 HT Sequence Detection system (Applied Biosystems).
MMP7 activity was determined using a fluorogenic MMP7 substrate assay (Merck Millipore, Nottingham, UK) and compared to negative control (chondrogenic medium) and positive control (recombinant human MMP7) according to the manufacturers' instructions.
The gene frequencies for controls were not in Hardy Weinberg equilibrium for the exon 3 polymorphism (P<0.0001) using the conventional polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) genotyping, whereas using a fluorogenic 5′nuclease TaqMan assay they were found to be in Hardy Weinberg equilibrium (P=0.93).
