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.


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


  • 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,
    see 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).

Table P17-42. SYBR® Green PCR Mix Selection Guide.

Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2)
KiCqStart® SYBR® Green qPCR ReadyMix™,
Cat. No. KCQS00
KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox ,
Cat. No. KCQS01
KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX,
Cat. No. KCQS02
KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ,
Cat. No. KCQS03
Compatible Instruments: Compatible Instruments: Compatible Instruments: Compatible Instruments:
Bio-Rad CFX384™ Applied Biosystems 7500 Applied Biosystems 5700 Bio-Rad iCycler iQ™
Bio-Rad CFX96™ Applied Biosystems 7500 Applied Biosystems 7000 Bio-Rad iQ™5
Bio-Rad MiniOpticon™ Fast Applied Biosystems ViiA 7 Applied Biosystems 7300 Bio-Rad MyiQ™
Bio-Rad MyiQ™ Stratagene Mx3000P® Applied Biosystems 7700  
Bio-Rad/MJ Chromo4™ Stratagene Mx3005P™ Applied Biosystems 7900  
Bio-Rad/MJ Opticon 2 Stratagene Mx4000™ Applied Biosystems 7900 HT Fast  
Bio-Rad/MJ Opticon®   Applied Biosystems 7900HT  
Cepheid SmartCycler®   Applied Biosystems StepOnePlus™  
Eppendorf Mastercycler® ep realplex   Applied Biosystems StepOne™  
Eppendorf Mastercycler® ep realplex2 s      
Illumina Eco qPCR      
Qiagen/Corbett Rotor-Gene® 3000      
Qiagen/Corbett Rotor-Gene® 6000      
Qiagen/Corbett Rotor-Gene® Q      
Roche LightCycler® 480      



Notes for this Protocol

Table P15-37. Potential Reference Gene Candidates and KiCqStart® Product Codes (KSPQ12012).


Reference Gene Mouse Human Rat
CANX M_Canx_1 H_CANX_1 R_Canx_1
HPRT1 NA H_HPRT1_1 R_Hprt1_1
PGK1 M_Pgk1_1 H_PGK1_1 R_Pgk1_1
TBP M_Tbp_1 H_TBP_1 R_Tbp_1
YWHAZ M_Ywhaz_1 H_YWHAZ_1 R_Ywhaz_1
ATP5B M_Atp5b_1 H_ATP5B_1 R_Atp5b_1
SDHA M_Sdha_1 H_SDHA_1 R_Sdha_1
TUBB2B NA H_TUBB2B_1 R_Tubb2b_1
UBC M_Ubc_1 H_UBC_1 R_Ubc_1
GUSB M_Gusb_1 H_GUSB_1 R_Gusb_1
TUBA1A NA H_TUBA1A_1 R_Tuba1a_1
ACTB M_Actb_1 H_ACTB_1 R_Actb_1



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.

Table P15-38. qPCR Master Mix for Reference Gene Selection.


Reagents Volume (μL) per Single
20 μL Reaction
2× KiCqStart® SYBR® Green qPCR ReadyMix™ 10
Forward primer (10 μM) 0.9
Reverse primer (10 μM) 0.9
PCR grade Water 3.2


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

Table P15-39. PCR Cycling Conditions for Reference Gene Selection.


Cycling Conditions Temp (°C) Time (sec)
Initial denaturation/Hot Start 95 30
Repeat steps 1–3 through 40 cycles
Step 1 95 5
Step 2 58 10
Step 3 72 15

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