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).

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

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).

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
PPIA	NA	H_PPIA_1	R_Ppia_1
GUSB	M_Gusb_1	H_GUSB_1	R_Gusb_1
GAPDH	NA	H_GAPDH_1	NA
TUBA1A	NA	H_TUBA1A_1	R_Tuba1a_1
ACTB	M_Actb_1	H_ACTB_1	R_Actb_1

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.

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. 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).