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qPCR Gene Expression/Copy Number Analysis Using SYBR Green I Dye Detection 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.

Measuring a target quantity relative to one or more stable reference genes using SYBR Green I dye detection is a common application of qPCR. Below is a standard protocol that can be adapted to specific experimental needs.

Experimental Objectives

After optimization of the qPCR assays for both target and reference genes, these are used to measure the quantity of target. A ratio is determined between the quantity of the gene of interest (GOI) and the stable reference gene(s) as described in Data Analysis. In this example, a standard curve is used for determination of copy number. However, a relative quantity can also be determined without a standard curve by using the alternative Comparative Quantification analysis method (see Data Analysis). If this approach is adopted, the standard curve is omitted but a calibrator sample is included alongside all test samples. Standard primer concentrations and annealing temperatures are included in the protocol but these should be adapted based on results from optimization experiments (see Primer Concentration Optimization and Primer Optimization Using Temperature Gradient).

Equipment

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

Reagents

  • gDNA 10ng to 100ng or cDNA to be used as template (diluted 1:2 for low expressed genes to 1:10 to 1:100 for medium to high expressed genes).
  • KiCqStart SYBR® Green ReadyMix™ (Sigma KCQS00/KCQS01/KCQS02/KCQS03—depends on instrument, refer
    to 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 genes (stock at 10 μM).

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

Notes for this Protocol

Method

1.    Prepare a different qPCR master mix for each primer pair to be run. Prepare sufficient mix for the samples, standard
       curve reactions (described as six dilutions below), No Template Controls, all in duplicate plus calculate an extra 10% to
       allow for pipetting error.

       For example, if there are five test samples, prepare a mix for samples (5×2) plus standard curve (6×2) plus No Template
       Control (NTC) (1×2) = 24 reactions. Therefore prepare a mix for 26.4 or 27 reactions per primer pair.

Table P17-43. Reaction Master Mix for SYBR Green I Detection.

 

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

 

2.    Prepare a 1:10 fold (or suitable dilution for the experiment) serial dilution of suitable standard curve template/cDNA so
       that there is 20 μL template of each dilution (six dilutions in total).

3.    Add 5 μL of appropriate template serial dilution (standard curve), sample test cDNA or water (NTC) to the defined
       tubes or wells.

4.    Add 15 μL of master mix to each tube or well.

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

6.    Run samples according to Table P17-44.

7.    See Data Analysis for guidance.

Table P17-44. PCR Cycling Conditions for Standard Curve Generation.

 

Cycling Conditions Temp (°C) Time (sec)
Denaturation/hot start 95 30
Steps 1–2 are repeated through 40 cycles
Step 1 95 5
Step 2 60 30

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

 

Materials