QPCR Troubleshooting

PROBLEM

CAUSES

SOLUTIONS

No qPCR Product or very low yield

Enzyme, master mix used improperly

• Use the master mixes as recommend, ensure the suggested protocol is followed.

Impurities inhibiting PCR

• Inhibitors from template, primers, and low-quality reagents can reduce yield. Purify template and use highest quality master mixes for qPCR.
• Do not use more than 10% of the reverse transcription reaction volume for the qPCR.

Faulty primer design, suboptimal primer concentration, low quality primers.

• Use dedicated software for primer design. Check primer quality on the denaturing polyacrylamide gel, check the PCR product on the agarose gel.
• Primers should always be present in excess in the reaction mixture and both at equal concentrations.
  
• Optimal primer concentration is always chosen empirically, too high primer concentration can reduce qPCR specificity due to increased misspriming.

 PCR product too long

• Design experiments for amplicons that are at least 500 bp. Best is about 150 bp.

Insufficient template or poor quality template.

• Repurify template and use check the quality and integrity on the agarose gel if possible. Increase template amount.

qPCR reagents containing fluorescent dyes or probe was exposed to light

• After setting up the reaction immediately return your reagents or probe to the product packaging to minimize exposure to light.

Setup errors

• Mix well all reagents before every use. Make mastermixes whenever possible as this reduces pipetting errors.

Component changes

• Monitor every change of the reagents or plastics used for qPCR. Every change may have influence on the results.

Suboptimal cycling

• Complete Initial DNA denaturation is essential to get good PCR yield. Typically 1-3min denaturation at 95°C is sufficient.

• During the cycling normally 30 sec DNA denaturation at 94-95°C per cycle is applied. For GC-rich DNA templates, this step could be prolonged by 5 sec increments.

• Primer Annealing temperature should be 5°C lower than the lowest primer melting temperature (Tm). The time of annealing is 30 sec. Too low annealing temperature leads to high background, too high one may lead to no product in PCR.

• The PCR extension stage is the primer elongation reaction performed typically at 72°C which is an optimal temperature for polymerase. Elongation stage should last 30 seconds for 500 bp amplicons.

Insufficient PCR cycles

• For low amount of template (10 copies and less) perform 40 cycles.

Short or long unspecific products

Faulty primer design, suboptimal primer concentration, low quality primers.

• Use dedicated software for primer design to avoid primer dimers.
• Check primer quality on the denaturing polyacrylamide gel, check the PCR product on the agarose gel.
• Primers should always be present in excess in the reaction mixture and both at equal concentrations.
  
• Optimal primer concentration is always chosen empirically, too high primer concentration can reduce PCR specificity due to increased misspriming.

Insufficient template or poor quality template.

• Purify genomic DNA carefully and avoid sharing. Use purification kits designed for the type of template DNA. Store purified template correctly, avoid too much mixing after thawing.
• Repurify the DNA if necessary and check the quality and integrity on the agarose gel if possible.
  
• Increase template amount.

Room temperature reaction setup

• Assemble the PCR reaction on ice, keep all reagents on ice.
  

Annealing temperature too low

• Primer Annealing temperature should be 5°C lower than the lowest primer melting temperature (Tm). The time of annealing is 30 sec.
• Too low annealing temperature leads to high background, too high one may lead to no product in qPCR.

Elongation too long

• The PCR extension stage is the primer elongation reaction performed typically at 72°C which is an optimal temperature for polymerase. Elongation stage should last 30 seconds for 500 bp amplicons. For shorter amplicons it should be shortened.

PCR contamination

• Keep a clean work environment. When preparing qPCR, care should be taken to eliminate the possibility of contamination with foreign DNA templates.
• Use separate clean areas for sample preparation, reaction mixture preparation and cycling.
• Before qPCR setup, clean your working space with 70% ethanol or with a special DNA-removing spray.
• Always wear fresh gloves.
• Use sterile tubes and pipette tips with aerosol filters for qPCR setup. Most contaminating templates come from dirty pipettes.
• Use DNA- and nuclease-free reagents, including water.
• Use the advantages of Uracil-DNA Glycosylase: Amplification of contaminating templates remaining in the work environment from previous experiments is one of the most common contamination problems. The use of Uracil-DNA Glycosylase (UDG or UNG) and dUTP (dNTP/dUTP Mix) helps prevent carry-over contamination.

Fluorescence in negative control

Contamination in reagents

• Change the reagents and consumables, if contamination is suspected.
  

Primer dimers

• Use dedicated software for primer design to avoid primer dimers.

Not reproducible results

Low quality regents

• Use high quality master mixes for qPCR and nuclease free water.
• Recheck the quality and integrity of the template before every qPCR if possible.
• Use only dedicated pipets and tubes of high quality plastic which is not binding the DNA or proteins.

Setup errors

• Mix well all reagents before every use. Make mastermixes whenever possible as this reduces pipetting errors.

Component changes

• Monitor every change of the reagents or plastics used for qPCR. Every change may have influence on the results.

Nuclease contamination

• Keep a clean work environment.
  
• Use separate clean areas for sample preparation, reaction mixture preparation and cycling.
• Before qPCR setup, clean your working space with 70% ethanol or with a special spray.
• Always wear fresh gloves.
• Use sterile tubes and pipette tips with aerosol filters for qPCR setup.
  
• Use DNA- and nuclease-free reagents, including water.