Luna® One-Step RT-qPCR Troubleshooting Guide

PROBLEM

PROBABLE CAUSE(S)

SOLUTION(S)

qPCR traces show low or no amplification

Reagent omitted from qPCR assay Reagent added improperly to qPCR assay

  • Verify all steps of the protocol were followed correctly

Incorrect cycling protocol

  • Refer to the proper qPCR cycling protocol in product manual

Incorrect channel selected for the qPCR thermal cycler

  • Verify correct optical settings on the qPCR instrument

DNA template or reagents are contaminated or degraded

  • Confirm the expiration dates of the kit reagents
  • Verify proper storage conditions provided in this user manual
  • Rerun the qPCR assay with fresh reagents
  • Confirm template input amount

Inconsistent qPCR traces for triplicate data

Improper pipetting during qPCR assay set-up

  • Ensure proper pipetting techniques

qPCR plate film has lost its seal, causing evaporation in the well. The resulting qPCR trace may show significantly different fluorescence values relative to its replicates

  • Ensure the qPCR plate is properly sealed before inserting into the qPCR thermal cycler.
  • Exclude problematic trace(s) from data analysis.

Poor mixing of reagents during qPCR set-up

  • Make sure all reagents are properly mixed after thawing them

Bubbles cause an abnormal qPCR trace

  • Avoid bubbles in the qPCR plate
  • Centrifuge the qPCR plate prior to running it in the thermal cycler
  • Exclude problematic trace(s) from data analysis

DNA standard curve has a poor correlation coefficient/ efficiency of the DNA standard curve falls outside the 90–110% range

Presence of outlying qPCR traces

  • Omit data produced by qPCR traces that are clearly outliers caused by bubbles, plate sealing issues, or other experimental problems

Improper pipetting during qPCR assay set-up

  • Ensure that proper pipetting techniques are used

Reaction conditions are incorrect

  • Verify that all steps of the protocol were followed correctly

Bubbles cause an abnormal qPCR trace

  • Avoid bubbles in the qPCR plate
  • Centrifuge the qPCR plate prior to running it in the thermal cycler

Poor mixing of reagents

  • After thawing, make sure all reagents are properly mixed

Threshold is improperly set for the qPCR traces

  • Ensure the threshold is set in the exponential region of qPCR traces
  • Refer to the real-time instrument user manual to manually set an appropriate threshold

Melt curve shows different peaks for low input samples

Non-template amplification is occurring

Infrequently, denaturation of a single species can occur in a biphasic manner, resulting in two peaks

  • Compare melt curve of NTC to samples
  • Redesign primers with a Tm of 60°C or use our Tm calculator to determine the optimal annealing temperature of the primers
  • Perform a primer matrix analysis to determine optimal primer concentrations

No template control qPCR trace shows amplification, NTC Cq is close to or overlapping lower copy standards

Reagents are contaminated with carried-over products of previous qPCR (melt curve of NTC matches melt curve of higher input standards)

  • Replace all stocks and reagents
  • Clean equipment and setup area with a 10% chlorine bleach
  • Consider use of 0.2 U/μl Antarctic Thermolabile UDG to eliminate carryover products

Primers produce non-specific amplification
(melt curve of NTC does not match melt curve of higher input standards)

  • Redesign primers with a Tm of 60°C or use qPCR primer design software