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
Incorrect reporter dye selected for the qPCR thermal cycler
  • 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
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

Amplification in No-RT control

RNA is contaminated with genomic DNA
  • Treat sample with DNaseI
  • Redesign amplicon to span exon-exon junction

 

Ordering Information

Product
NEB #
Size
Luna Universal One-Step RT-qPCR Kit E3005S/L 200/500 reactions
Luna Universal One-Step RT-qPCR Kit E3005X 1,000 reactions
Luna Universal One-Step RT-qPCR Kit E3005E 2,500 reactions
Companion Products
Antarctic Thermolabile UDG M0372S/L 100/500 units
Luna Universal Probe One-Step RT-qPCR Kit E3006S/L 200/500 reactions
Luna Universal Probe One-Step RT-qPCR Kit E3006X 1,000 reactions
Luna Universal Probe One-Step RT-qPCR Kit E3006E 2,500 reactions
Luna Universal qPCR Master Mix M3003S/L 200/500 reactions
Luna Universal qPCR Master Mix M3003X 1,000 reactions
Luna Universal qPCR Master Mix M3003E 2,500 reactions
Luna Universal Probe qPCR Master Mix M3004S/L 200/500 reactions
Luna Universal Probe qPCR Master Mix M3004X 1,000 reactions
Luna Universal Probe qPCR Master Mix M3004E 2,500 reactions
LunaScript RT SuperMix Kit E3010S/L 25/100 reactions