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

PCR Technologies Protocols Table of Contents

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 (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 (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™ (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

• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)
• PCR tubes and plates, select one to match desired format:
  •    Individual thin-walled 200 μL PCR tubes (Z374873 or P3114)
  •    Plates
        - 96-well plates (Z374903)
        - 384-well plates (Z374911)
  •    Plate seals
        - ThermalSeal RTS™ Sealing Films (Z734438)
        - ThermalSeal RT2RR™ Film (Z722553)

Notes for this Protocol

• cDNA is generated using random priming or oligo-dT method (Standard Reverse Transcription Protocol (Two-step)).
• Dilute forward and reverse primers to 10 μM or to an appropriate concentration determined as a result of optimization (Primer Concentration Optimization and Primer Optimization Using Temperature Gradient).
• If using a PCR plate, follow a plate schematic to ensure that the reaction mix, samples and controls are added to the correct wells.
• All tests will be run as duplicate reactions.

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.

 

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).