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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™ (KCQS00/KCQS01/KCQS02/KCQS03
  • 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). 
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 1.SYBR® Green PCR Mix™ Selection Guide.

Supplies

Notes for this Protocol

  • cDNA is generated using random priming or oligo-dT method (see Standard Reverse Transcription Protocol (Two-step)). The RT product is diluted to prepare the standard curves (1:2 and 1:10 are given as examples). RNA can also be diluted and cDNA synthesized from each dilution using the ReadyScript® kit or a one-step RT-qPCR approach can be adopted by diluting RNA and following the one-step RT-qPCR approach in Reverse Transcription Protocol (One-step SYBR® Green I Dye Detection) and Reverse Transcription Protocol (One-step Probe Detection). Alternatively, DNA templates can be substituted such that the expected Cq range is within Cq 15 to Cq 38.
  • Each concentration of the serial dilutions will be run as duplicate reactions.

Method

1.    Prepare a qPCR master mix that is sufficient for 40 reactions following Table 2. This allows for extra to
       accommodate pipetting errors since 32 reactions will be run (Table 3).

ReagentsVolume (μL) per
Single 20 μL Reaction
Volume (μL) for
40 Reactions
2× KiCqStart® SYBR® Green qPCR
ReadyMix™
10400
Forward primer (10 μM)0.936
Reverse primer (10 μM)0.936
PCR grade water3.2128
Table 2.Reaction Master Mix for Generation of 1:2 and 1:10 Standard Curve.

2.    Dilute the DNA through a series of 1:10 and 1:2 covering 7 dilution points for each series (Table 3, Plate Layout for DNA Dilution).

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

Plate Layout for DNA Dilution
Plate
Column
1234Rest of Plate
A1111 
B0.10.10.50.5 
C0.010.010.250.25 
D0.0010.0010.1250.125 
E0.00010.00010.06250.0625 
F0.000010.000010.031250.03125 
G0.0000010.0000010.0156250.015625 
HNTCNTCNTCNTC 
Table 3. 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).

4.    Add 15 μL of master mix to each well (Table 3).

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.

Cycling ConditionsTemp (°C)Time (sec)
Initial denaturation/Hot Start9530
Steps 1–2 are repeated through 40 cycles
Step 1955
Step 26030
Table 4.PCR Cycling Conditions for Standard Curve Generation.

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

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