I am setting up a new StepOne Real-Time PCR system (48 well) that came with a laptop computer with all the installation completed. The RNase P Experiment "Failed". There were virtually no significant outliers, therefore trying to screen out any seemingly outliers did not resolve the problem and the . The R2 value was 0.956. I have no other RNase P plate to repeat the RNase P verification experiment. Does this mean I cannot use the instrument with my own experiments until I have obtained a new plate and passed the verification test? What might be the reasons for the failed verification experiment?
Alternative to Nilima, if you have the abolity to perform experiments in another istrument (Real-Time), you should do a reaction for 2 housekeeping genes in parrarel and compare the data.
However, these plates are only for verification. The calibration is performed by the manufactures.
Hi, just today guy from Abi came to install the step one plus at our faculty. He said that, if the RNase P test failed, the most probably it was due to human error (for example, the samples in plate were not completely down or you had bubble in some wells e.t.c.) or it might be that some particle fell into some of the wells (but then I suppose you would have outliers). However, he said that in that case we should reorder the RNase P plate (normally without paying for the kit because it is not our fault), and if this does not work, than we have to call the maintenance staff. So, yes, i think you should wait for the new kit to arrive...
I have used the StepOne under challenging files conditions, including during the influenza A/H1N1 pandemic in 2009. Though I haven't any experience with the RNAase P experiment, we obviously had to run a reverse transcriptase step to ake our detection of RNA virus targets possible. I remember we had a bit of crosstalk between FAM and ROX detection, which we resolved by adjusting the respective thresholds manually. I'll share the question with some of my colleagues.
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