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qPCR Efficiency Determination 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.

Once an assay has been optimized, it is important to verify the reaction efficiency. This information is important when reporting and comparing assays. In this example protocol, the assay efficiency is compared over a wide and narrow dynamic range of cDNA concentrations. In practice, it is common to select a single range to test depending on the expected range of target in the samples, so the protocol given can be adjusted according to the requirements of the experiment. In this example the efficiency is calculated using both 10-fold and 2-fold dilution series. The standard curve should encompass the expected range of expression for the genes of interest Note that the proportionality of the cDNA yield with respect to RNA input is linear when using the ReadyScript® RT kit, so this experiment can be adapted to RT-qPCR if using that system by 1) diluting the RNA and running the RT reactions and 2) then running qPCR on each of the resulting cDNA samples (see Reverse Transcription for examples).  However, this is not always the case and does not apply for all Reverse Transcription kits or protocols. This should be verified before adapting this protocol to an alternative kit

Equipment

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

Reagents

  • DNA to be used as the standard curve template (e.g., cDNA, gDNA or a synthetic template).
  • KiCqStart SYBR® Green ReadyMix™ (Sigma 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 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 qPCR master mix that is sufficient for 40 reactions following Table P16-40. This allows for extra to
       accommodate pipetting errors since 32 reactions will be run (Table P16-41).

Table P16-40. Reaction Master Mix for Generation of 1:2 and 1:10 Standard Curve.

 

Reagents Volume (μL) per
Single 20 μL Reaction
Volume (μL) for
40 Reactions
2× KiCqStart SYBR Green qPCR
ReadyMix
10 400
Forward primer (10 μM) 0.9 36
Reverse primer (10 μM) 0.9 36
PCR grade water 3.2 128

 

2.    Dilute the DNA through a series of 1:10 and 1:2 covering 7 dilution points for each series (see Table P16-41,
       Plate Layout for DNA Dilution).

3.    Add 5 μL of appropriate template dilution to the defined wells (see Table P16-41, Plate Layout for DNA Dilution).

Table P16-41. Diagram of the First Four Columns of a 96-well Plate Layout Showing the Position of Standard Curve Template Dilutions. Dilution stated is relative to the original stock solution. When using an artificial template it is possible to calculate copy number (relative to OD readings).

 

Plate Layout for DNA Dilution
Plate
Column
1 2 3 4 Rest of Plate
A 1 1 1 1  
B 0.1 0.1 0.5 0.5  
C 0.01 0.01 0.25 0.25  
D 0.001 0.001 0.125 0.125  
E 0.0001 0.0001 0.0625 0.0625  
F 0.00001 0.00001 0.03125 0.03125  
G 0.000001 0.000001 0.015625 0.015625  
H NTC NTC NTC NTC  

 

4.    Add 15 μL of master mix to each well (see Table P16-41).

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

6.    Run samples according to the two-step protocol below. Steps 1–2 are repeated through 40 cycles. Follow
       amplification with a standard dissociation curve analysis.

Table P16-42. PCR Cycling Conditions for Standard Curve Generation.

 

Cycling Conditions Temp (°C) Time (sec)
Initial 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