qPCR Reference Gene Selection Protocol

Optimization of qPCR Conditions

Optimization of qPCR conditions is important for the development of a robust assay. Indications of poor optimization are a lack of reproducibility between replicates as well as inefficient and insensitive assays. The two main approaches are optimization of primer concentration and/or annealing temperatures.

Analysis of gene expression data requires a stable reference or loading control. This reference is usually one or more reference genes. Suitable reference genes are those which are unaffected by differences in samples and experimental treatments and these must be determined for each experimental model. When performing a trial to select stable reference genes it is critical that the genes selected are from different biological pathways and that their expression is independently regulated. Ideally all reference gene candidates are tested on a selection of five test and five control samples.

Equipment

• Quantitative PCR instrument
• Laminar flow hood for PCR set up (optional)

Reagents

• cDNA diluted 1:100 (the more dilute cDNA is usually sufficient to detect highly expressed reference genes, for medium expressed genes use 1:10 dilution).
• KiCqStart^® SYBR^® Green ReadyMix™ (KCQS00/KCQS01/KCQS02/KCQS03—depends on instrument, Table P4-6).
• PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
• Forward and reverse primers for test reference genes (stock at 10 μM). Note: a suitable list of genes is provided in Table P15-37. These are available as KiCqStart^® Primers, which are pre-designed assays as shown (the sequences of the primers are provided with product delivery).

Supplies

• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)
• PCR tubes and plates, select one to match desired format:
  • Individual thin-walled 200 μL PCR tubes (Z374873 or P3114)
  • Plates
  - 96-well plates (Z374903)
  - 384-well plates (Z374911)
  • Plate seals
  - ThermalSeal RTS™ Sealing Films (Z734438)
  - ThermalSeal RT2RR™ Film (Z722553)

Notes for this Protocol

• cDNA is generated using ReadyScript^® RT kit incorporating a combination of random and oligo-dT priming (see Standard Reverse Transcription Protocol (Two-step) and Reverse Transcription).
• If using a PCR plate, follow a plate schematic to ensure that the reaction mix, samples and controls are added to the correct wells.
• Test a wide range of genes that may be potential reference genes. These should be expressed in different functional gene pathways. A selection of potential KiCqStart^® SYBR^® Primers for some model organisms is given in
  Table P15-37. OligoArchitect is a suitable design tool for alternative primer designs (see OligoArchitect™ Assay Design and PCR/qPCR/dPCR Assay Design).

Method

1. Calculate the number of reactions required for each reference gene. Prepare sufficient mix for two reactions per
sample. For example if testing 5 test and 5 control samples, two No Template Controls (NTC) = 22 reactions. Calculate
sufficient for 10% extra to allow for pipetting error.

2. Prepare qPCR master mix for each Reference Gene primer pair according to Table P15-38. Do not add cDNA to the
master mix. Mix well and avoid bubbles.

3. Add 15 μL of master mix to the defined tubes/wells.

4. Add 5 μL of appropriate template (sample or water for NTC to the defined tubes/wells).

5. Cap tubes or seal plates and label. (Make sure the labeling does not obscure instrument excitation/detection light path.)

6. Run reactions according to the three-step protocol below (Table P15-39). Steps 1–3 are repeated through 40 cycles.

7. Data Analysis to analyze data and determine the most stable reference gene or combination of genes.

Note: Use standard dissociation curve protocol (data collection).