[Skip to Content](https://www.sigmaaldrich.com#main-content) [![MilliporeSigma](https://www.sigmaaldrich.com/static/logos/purple/millipore_sigma.svg)](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[qPCR](https://www.sigmaaldrich.com/US/en/applications/genomics/qpcr)qPCR Reference Gene Selection Protocol # qPCR Reference Gene Selection Protocol ## [PCR Technologies Protocols Table of Contents](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/pcr-technologies-protocols-introduction) ## 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. [](https://www.sigmaaldrich.com/US/en/product/sigma/W1754)[](https://www.sigmaaldrich.com/US/en/product/sigma/W1754) 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](https://www.sigmaaldrich.com/US/en/product/sigma/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](https://www.sigmaaldrich.com/US/en/product/sigma/KSPQ12012), 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](https://www.sigmaaldrich.com/US/en/product/sigma/KCQS00)KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox , [Cat. No. KCQS01](https://www.sigmaaldrich.com/US/en/product/sigma/KCQS01)KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX, [Cat. No. KCQS02](https://www.sigmaaldrich.com/US/en/product/sigma/KCQS02)KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ, [Cat. No. KCQS03](https://www.sigmaaldrich.com/US/en/product/sigma/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[](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/standard-reverse-transcription-protocol-two-step)[](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/standard-reverse-transcription-protocol-two-step) - Sterile filter [pipette tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) - [Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)](https://www.sigmaaldrich.com/US/en/product/sigma/CLS430909) - PCR tubes and plates, select one to match desired format: •    Individual thin-walled 200 μL PCR tubes (Z374873 or [P3114](https://www.sigmaaldrich.com/US/en/product/sigma/P3114)) •    Plates       - [96-well plates (Z374903)](https://www.sigmaaldrich.com/US/en/product/sigma/Z374903)       - [384-well plates (Z374911)](https://www.sigmaaldrich.com/US/en/product/sigma/Z374911) •    Plate seals       - [ThermalSeal RTS™ Sealing Films (Z734438)](https://www.sigmaaldrich.com/US/en/product/sigma/Z734438)       - [ThermalSeal RT2RR™ Film (Z722553)](https://www.sigmaaldrich.com/US/en/product/sigma/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)](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/standard-reverse-transcription-protocol-two-step) and [Reverse Transcription](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/gene-expression-and-silencing/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 f](https://www.sigmaaldrich.com/US/en/product/sigma/KSPQ12012)or some model organisms is given in  __Table P15-37__. OligoArchitect is a suitable design tool for alternative primer designs (see [OligoArchitect™ Assay Design](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/qpcr/oligoarchitect-assay-design) and [PCR/qPCR/dPCR Assay Design](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/pcr-qpcr-dpcr-assay-design)). | | | | | |----------------|-------------|--------------|--------------| | 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 | Table P15-37Potential Reference Gene Candidates and KiCqStart® Product Codes [(KSPQ12012).](https://www.sigmaaldrich.com/product/sigma/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](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/qpcr/data-analysis) to analyze data and determine the most stable reference gene or combination of genes. | | | | |----------------------------------------|-----------|------------| | 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 | Table P15-39PCR Cycling Conditions for Reference Gene Selection. __*Note:*__ Use standard dissociation curve protocol (data collection). ## Materials Sorry, an unexpected error has occurred Response not successful: Received status code 500 __Related Articles__ - [FFPE Tissue](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/wga1-ffpe) - [MystiCq® MicroRNA® Quantitation System](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/gene-expression-and-silencing/mysticq-microrna-analysis) - [Multiplex Real-Time PCR](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/kapa-multiplex-rtpcr) - [Protocol for Conjugating NHS-Ester Modifications to Amino-Labeled Oligonucleotides](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/nhs-ester-oligonucleotide-conjugation) - [Reverse Transcription Protocol (One-step Probe Detection)](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/pcr/reverse-transcription-one-step-probe-detection) - [The 3'/5' Assay for Analysis of RNA Integrity Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/qpcr/3-5-assay-for-analysis-of-rna-integrity) - [ChIP-qPCR Data Analysis](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/qpcr/chip-qpcr-data-analysis) - [dPCR Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/qpcr/dpcr) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Sealing Films, Foils & Tapes](https://www.sigmaaldrich.com/US/en/products/labware/sealing-films-foils-and-tapes) - [Pipette Tips](https://www.sigmaaldrich.com/US/en/products/labware/liquid-handling-and-dispensing/pipette-tips) - [Allergenic Fragrance Testing – Certified Materials](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/allergenic-fragrance-testing) - [Analysis of Alkylphenols Using New Internal Standards](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/analysis-of-alkylphenols) - [FAME Standard for Optimizing GC System Performance](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/fame-standard-for) - [Fast and Reliable Environmental Analysis of Aldehyde and Ketone Air Pollutants](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/fast-and-reliable) - [Hydrocarbon Oil Index in Samples](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/hydrocarbon-oil-index) - [Neonicotinoids](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/neonicotinoids-bee-pesticides) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) Top __Sign In To Continue__ To continue reading please sign in or create an account. 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