User manual

Table of Contents
Droplet Digital
™
PCR Applications Guide | iv
Statistics of ddPCR .............................................................30
Copies per Microliter .........................................................31
Copies per Droplet ...........................................................31
Low Concentration Example ......................................................32
Intermediate Concentration Example ................................................33
High Concentration Example ......................................................33
Looking across the Whole Concentration Range .......................................34
Concentration Calculation ........................................................34
Definitions .................................................................34
Formula for Calculating Concentration ............................................35
Derivation of Concentration Formula ..............................................35
Errors in ddPCR ................................................................36
Chapter 4 Copy Number Variation Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
Overview .....................................................................37
CNV Calculations ...............................................................39
CNV Analysis in Homogeneous Samples .............................................39
CNV Analysis in Heterogeneous Samples ............................................39
Planning CNV Experiments .......................................................40
Assay Design ...............................................................40
Running a CNV Assay ........................................................41
Restriction Digestion ..........................................................41
DNA Loading for Lower-Order CN Analysis (diploid CN <10) ...........................44
DNA Loading for Higher-Order CN Analysis (diploid CN >10) ...........................44
Chapter 5 Rare Mutation and Sequence Detection ...........................................45
Overview .....................................................................45
Rare Mutation Detection .........................................................47
ddPCR for Rare Allele Detection and Experimental Considerations .......................47
RMD Experiment Considerations ................................................48
Testing an RMD Assay ........................................................48
Interpreting 2-D Plot Results for SNP Assays .......................................50
Statistical Considerations for Rare Detection Experimental Design .......................51
Recommended Controls .........................................................52
Experimental Strategies for RMD ................................................53
Factors that Impact RMD Calculations ............................................53
Sample Preparation ..........................................................53
Rare Sequence Detection ........................................................53
RSD Experimental Strategies ...................................................54
Case 1: Quantification with Respect to Total Starting Volume ...........................54
Case 2: Quantification with Respect to Second DNA Sequence .........................55
Factors that Impact RSD Calculations ............................................56
References ...................................................................56
Chapter 6 Gene Expression ..............................................................57
Overview .....................................................................57
Two-Step Reverse Transcription ddPCR .............................................58
Obtain RNA ................................................................58
Generate cDNA .............................................................58