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HomeqPCRqPCR Reference Gene Selection Protocol

qPCR Reference Gene Selection Protocol

PCR Technologies Protocols Table of Contents

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).
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 7500Applied Biosystems 5700Bio-Rad iCycler iQ™
Bio-Rad CFX96™Applied Biosystems 7500Applied Biosystems 7000Bio-Rad iQ™5
Bio-Rad MiniOpticon™Fast Applied Biosystems ViiA 7Applied Biosystems 7300Bio-Rad MyiQ™
Bio-Rad MyiQ™Stratagene Mx3000P®Applied Biosystems 7700
Bio-Rad/MJ Chromo4™Stratagene Mx3005P™Applied Biosystems 7900
Bio-Rad/MJ Opticon 2Stratagene 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
Table P17-42SYBR® Green PCR Mix Selection Guide.

Supplies

Notes for this Protocol

Reference GeneMouseHumanRat
CANXM_Canx_1H_CANX_1R_Canx_1
HPRT1NAH_HPRT1_1R_Hprt1_1
PGK1M_Pgk1_1H_PGK1_1R_Pgk1_1
TBPM_Tbp_1H_TBP_1R_Tbp_1
YWHAZM_Ywhaz_1H_YWHAZ_1R_Ywhaz_1
ATP5BM_Atp5b_1H_ATP5B_1R_Atp5b_1
SDHAM_Sdha_1H_SDHA_1R_Sdha_1
TUBB2BNAH_TUBB2B_1R_Tubb2b_1
UBCM_Ubc_1H_UBC_1R_Ubc_1
PPIANAH_PPIA_1R_Ppia_1
GUSBM_Gusb_1H_GUSB_1R_Gusb_1
GAPDHNAH_GAPDH_1NA
TUBA1ANAH_TUBA1A_1R_Tuba1a_1
ACTBM_Actb_1H_ACTB_1R_Actb_1
Table P15-37Potential Reference Gene Candidates and KiCqStart® Product Codes (KSPQ12012).

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.

ReagentsVolume (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 Water3.2
Table P15-38qPCR Master Mix for Reference Gene Selection.

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.

Cycling ConditionsTemp (°C)Time (sec)
Initial denaturation/Hot Start9530
Repeat steps 1–3 through 40 cycles
Step 1955
Step 25810
Step 37215
Table P15-39PCR Cycling Conditions for Reference Gene Selection.

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

Materials
Product No.Product NameDescriptionPricing
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