Plexor HY: inconsistent results? - (Nov/18/2008 )
Hi everyone,
we are currently testing the Plexor HY kit from Promega and we have obtained so far very odd results.
We realized dilution series starting from DNA quantified with different methods and the results obtained with Plexor are between 3 and 5 fold higher than expected.
Moreover we used some highly inhibited samples and the Ct value of the internal PCR control is not different than the one obtained with non inhibited samples.
Is anyone here has ever tried this kit?
and if yes, did you face the same problem?
Thank you in advance!
We realized dilution series starting from DNA quantified with different methods and the results obtained with Plexor are between 3 and 5 fold higher than expected.
1. Were the comparisons made with the same template? If yes, did you re-quantify the template on the day of Plexor® HY quantitation? Long term storage can change DNA concentration.
2. The target mass of DNA required for each system can differ. This needs to be mathematically calculated. Most often the Validation Guide for that product will address that. You should contact the vendor to obtain the guide. Try the link below. Validation Guide for the Plexor® HY System
Moreover we used some highly inhibited samples and the Ct value of the internal PCR control is not different than the one obtained with non inhibited samples.
What kind of inhibitor are you using? Did you try several concentrations of the inhibitor?
1. Were the comparisons made with the same template? If yes, did you re-quantify the template on the day of Plexor® HY quantitation? Long term storage can change DNA concentration.
We used a fresh DNA extract for the dilution series and the same template with Quantifiler and the spectrometer and the 2 different Taqman assays.
2. The target mass of DNA required for each system can differ. This needs to be mathematically calculated. Most often the Validation Guide for that product will address that. You should contact the vendor to obtain the guide. Try the link below. Validation Guide for the Plexor® HY System
I'll check that and see what we can take out of it.
Moreover we used some highly inhibited samples and the Ct value of the internal PCR control is not different than the one obtained with non inhibited samples.
What kind of inhibitor are you using? Did you try several concentrations of the inhibitor?
We used live samples, most likely the inhibitors are humic acid. But Promega told us that sometimes inhibitors are not detected because it seems that the new taq they are using is too robust...
Anyway we will amplify these samples and see what the results look like taking inot consideration the results of Plexor. therefore we will see if there is any correlation between these results and the amplification