1. Introduction to qPCR 

1.1 What is qPCR and how does it work? 4

2. Detection methods in qPCR 

2.1 Dye-based detection 6

2.2 Melt curve analysis 7

2.3 Probe-based detection 8

3. Target choice, primer and probe design 

3.1 Target 12

3.2 Primer design 12

3.3 Probe design 12

3.4 Multiplex qPCR design 14

4. Primer validation and reaction efficiency calculation 

5. Quantification methods in qPCR 

5.1 Absolute quantification 18

5.2 Relative quantification 19

6. Experimental setup, data analysis and MIQE 

6.1 Setting up qPCR experiments 21

6.2 Analysis and peer-review quality data reporting 22

6.3 Absolute quantification data analysis 22

6.4 Relative quantification data analysis 23

6.5 Data reporting 24

7. Applications 

7.1 1-Step RT-qPCR and 2-Step RT-qPCR 27

7.2 Gene expression 27

7.3 Species/allele abundance 28

7.4 Diagnostics 28

7.5 Genotyping 29

7.6 High Resolution Melt (HRM) curve analysis 29

8. Guidelines for testing qPCRBIO products 

8.1 General comments 31

8.2 Setting up test experiments 31

8.3 Cycling 32

8.4 Data analysis 32

9. Troubleshooting 

9.1 Issues with dye-based qPCR 35

9.2 Issues with probe-based qPCR 38

9.3 Issues with reverse transcription 41

10. References 

11. Disclaimer