qPCR

INTRODUCTION

Quantitative PCR (qPCR), also known as Real Time PCR, or Kinetic PCR is a quantitative version of the standard PCR reaction. qPCR not only indicates whether a particular DNA sequence is present in a sample (i.e qualitative) it also delivers exactly how much of a particular sequence is present relative to other samples and/or to standards (i.e quantitative).

NOTE: qPCR is sometimes confused with Reverse Transcription PCR due to the fact that both Reverse Transcription PCR and Real Time PCR can be abbreviated to RT-PCR. Strictly speaking the abbreviation RT-PCR always refers to Reverse Transcription PCR, Quantitative or Real Time PCR should always be abbreviated qPCR.

The key difference between the standard PCR reaction and qPCR is that while PCR waits until the end of the PCR reaction (all 30 + temperature cycles) before analyzing the amount of PCR product, qPCR takes a measurement at the end of each temperature cycle. In order to take this measurement a specialized qPCR instrument is needed such as those listed in table 1. The following protocol is applicable to all of these instruments (as well as others), though you should consult the instrument manufacturers recommendations and make amendments for differences in volume or the addition of other specific components in the master mix.

MATERIALS NEEDED

• CoolBox 2XT PCR ice-free cooling workstation or, alternatively, CoolRack PCR and CoolRack M15 modules with ice pan

• Vortexer

• Microcentrifuge tubes

• Centrifuge (temperature controlled, >3,000 x g)

• qPCR instrument (see table 1 below)

Manufacturer Instrument
ABI StepOne, StepOne Plus, PRISM®7000, 7500, 7500 Fast, 7700, 7900HT, ViiA 7 System, QuantStudio 12K Flex system

Roche

LightCycler® 96, and 480

Cepheid


Smart Cycler®, Gene Expert
Bio-Rad CFX96 Touch, CFX384 Touch, CFX Connect, CFX96 Touch Deep Well, Mini Opticon, CFX Automation
MJ Research (now part of Bio-Rad) DNA Engine Opticon™, Opticon® 2, and Chromo 4™ Real-Time Detector,
Agilent (Stratagene) Mx3000P®, Mx3005P™, and Mx4000®

Eppendorf

Mastercycler® ep realplex, Mastercycler pro

 

Table 1. Above lists some of the more popular qPCR instruments, included here are the current offering from each of the manufacturers together with some of the legacy systems, which while no longer available for purchase may still be in use in the laboratory. The protocol provided below is suitable for use on any of these instruments, as well as other systems not listed here. Always refer to the instructions for your specific instrument for any special requirements.

The accuracy of qPCR is highly dependent on set up. Accurate pipetting and proper mixing of solutions, is imperative. Extra care needs to be taken when pipetting, and thorough vortex mixing is mandatory. Retain all samples, master mix, and reaction tubes/plate on the appropriate CoolBox 2XT PCR workstation to ensure consistent controlled temperature or < 4°C and uniform conditions for all samples. This eliminates problems due to evaporation, degradation of reaction components, non-specific binding, and amplification during set up.

NOTE: Even Hot-start Polymerase can potentially exhibit a low level of amplification at room temperature. In fact, setting up a Hot-start reaction on ice is referred to as ‘pseudo hot-start’ protocol and is often used to trouble shoot qPCR problems. Pseudo hot start is frequently not used as a front line technique due to the potential for contamination, mess, and additional work that is involved working with ice. The Coolbox 2XT workstation however eliminate these problems, making pseudo hot-start as convenient as true hot start and enables use of pseudo hot start as a best practice when setting up qPCR.

For ease of set up as well as optimal results we have used KAPA SYBR FAST qPCR master mix in the following set up. This mix includes all of the required ingredients except for DNA template and primers. The polymerase used is KAPA SYBR DNA Polymerase which has been engineered to perform optimally under stringent qPCR reaction conditions.

INSTRUCTIONS

The following quantities are for a 20μL reaction volume. Quantities can be scaled for 10μL, or other reaction volumes as required.

  1. 1. Place the CoolBox Cooling Cartridge and CoolRack modules in the -20°C or -80°C freezer overnight prior to starting the qPCR set-up.

NOTE: Once removed from the freezer, the Coolbox workstation will provide stable and controlled temperature of < 4°C for over 10 hours.

  1. 2. Create a master mix of reagents. The volumes below are per sample, e.g., for 10 samples multiply each component by 10, plus a 10% extra. Calculate the required volumes of each component based on the following table. Once you have added all of the ingredients together, vortex gently to mix, and retain on ice while you prepare the DNA samples. Add the reagents in the order given in the table:

 

Ingredient

Quantity
PCR grade DI water As required to achieve a 20μl final reaction volume (including the DNA sample)
qPCR Master Mix (2X) e.g., KAPA SYBR FAST Universal Master Mix 10 µL
Forward Primer (10 μM) 200 nM (0.4 μL)
Reverse Primer (10 μM) 200 nM (0.4 μL)

 

  1. 3. Determine the appropriate volume of template DNA based on the concentration of your samples. Each reaction will require 20ng/20μL reaction volume)
  2.  

  3. 4. Transfer the appropriate volume of reaction mixture to each well of a PCR tube/plate.

     

  4. 5. Carefully add template DNA to each well
  5.  

  6. 6. Cap or seal the reaction tube/plate and centrifuge briefly.
  7.  

  8. 7. Take reaction plate/tubes from the CoolBox 2XT and transfer onto the qPCR instrument
  9.  

  10. 8. Run the qPCR protocol following manufacturers instructions and analyze results accordingly.
  11.  

See other protocols:

BioCision RNA isolation protocol
BioCision RT-PCR protocol
BioCision General PCR application
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Learn more about:

CoolRack tube modules

CoolBox XT and CoolBox 2XT ice-free cooling workstations

Ice pans and buckets

TruCool ergonomic microfuge tubes

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