Troubleshooting Guide: mRNA Quantitation Kits

Observation: High background level

Problem	Corrective Action
Insufficient washing	Wash per protocol being sure to remove all Wash Buffer from wells before addition of next component
Contamination with alkaline phosphatase	Keep work area clean and free of alkaline phosphatase

Poor precision (high coefficient of variation) for samples or calibrator

Problem	Corrective Action
Plate not washed before use	Wash per protocol
RNase contamination	Use RNase-free technique
Pipetting error	Use a new pipet tip for each pipetting step and use proper technique

Observation: No Signal for calibrator curve

Problem	Corrective Action
Component or step omitted	Read protocol thoroughly before repeating the assay
RNase contamination	Use RNase-free technique

Observation: Sample signal is above calibrator curve range

Problem	Corrective Action
Too much sample used	Repeat assay using less sample

Observation: Sample signal is below calibrators curve range, but calibrator curve works

Problem	Corrective Action
Samples may be degraded	Run RNA get to assess sample integrity
Inadequate amount of sample used	Repeat assay using more sample
Samples do not contain target mRNA	Verify that cell source was harvested near peak level of target mRNA