Luna® One-Step RT-qPCR Troubleshooting Guide

OBSERVATION

PROBABLE CAUSE(S)

SOLUTION(S)

qPCR traces show low or no amplification

Incorrect RT step temperature or RT step omitted

• For typical use, a 55°C RT step temperature is optimal for the Luna WarmStart Reverse Transcriptase (see cycling protocol).

Cycling protocol is otherwise incorrect

• Refer to the proper RT-qPCR cycling protocol in this user manual

Reagent omitted from RT-qPCR assay

Reagent added improperly to RT-qPCR assay

• Verify all steps of the protocol were followed correctly

• Select FAM/SYBR on the qPCR instrument

RNA template or reagents are contaminated or degraded

• Prepare high quality RNA without RNAse/DNase contamination
• Confirm template input amount
• Confirm the expiration dates of the kit reagents Verify proper storage conditions provided in this user manual
• Rerun the RT-qPCR assay with fresh reagents

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

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

Cycling protocol is incorrect

• Refer to the proper RT-qPCR cycling protocol in this user manual
• Use a 55°C RT step temperature
• For ABI instruments, use a 1 minute 60°C annealing/extension step

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 RT-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

• 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 C_q 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 or use products already containing UDG (e.g., NEB #M3019, L4001)

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

Amplification in No-RT control

• Treat sample with DNaseI
• Redesign primer to span exon-exon junction