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In this episode, we discuss: Droplet Digital PCR in Cell and Gene Therapy
- Robust methodology for viral titer using ddPCR
- Precise copy number determination and improved confidence in acceptance criteria
- Enhance your throughput and minimize hands-on time
- Learn how ddPCR is used in other key applications
00:10 Introduction
3:23 What is ddPCR and why is it considered a crucial technology in cell and gene therapy?
5:23 I currently use ddPCR for AAV viral titer, could it be used for other applications?
8:12 When designing duplex experiments, what considerations need to be made?
10:44 Situation: You merge the data from two replicate wells that have very different concentrations (for example, pipetting error). Question: Are there outputted statistics that tell you the confidence of the merged result?
12:50 I use regular TaqMan qPCR for several applications. My question is, is there any criteria or minimum requirement to apply for ddPCR?
15:11 How do I improve separation of positive and negative droplets?
17:40 I use ddPCR to titrate AAV. I was recommended (by a local BioRad rep) to run 50 cycles routinely to get cleaner results. But, in a recent webinar, it was strongly suggested not to exceed 45 cycles. What is your opinion?
20:04 Situation: You have multiple sets of primers/probe you are testing against a Gene of Interest. After 40 cycles of thermocycling, they appear equivalent. Question: Are there any other experiments you could run to help identify the “best” primers/probe set?
22:44 Is there a recommended diluent for rAAV samples prior to ddPCR?
24:22 When using two channels for ddPCR, my result will then be the average of the reading of those?
26:37 When you want to quantify viral genomes in tissues, the ratio to the reference gene represents the viral genomes/host genome?
28:10 What is the minimum number of droplets that can be called 'positive'? For example, if you have 4 positive droplets — is that enough, or is there a threshold that must be achieved?
31:51 Do you recommend adding BSA to the reaction?
33:17 For multiplexing, can the Quantsoft Analysis Pro [Software] gate populations automatically or does it always have to be manual?
34:34 If you have less than 5 copies per microliter using qPCR to test viral load in tissue samples, would you recommend ddPCR to detect the amount of virus in these samples?
36:45 For quantification of the level of DNA methylation at a target region, would you recommend using probes complementary to the methylated and unmethylated versions of the target sequence for the 2 channels in one well?
38:22 Why is hotstart not typically recommended for ddPCR? I'm sure this is based on multiple data comparisons, but while selecting primers and probes, is it recommended to compare hotstart versus not?
40:15 When using two channels for ddPCR, my result will then be the average of the reading of those?
43:58 Let's say there are 2 primer/probe, and one is sensitive than another. Does the sensitivity of primer/probe affect the result of ddPCR?
47:25 Is ddPCR mostly used for research purposes? Are there any routine clinical applications for ddPCR?
49:49 What are the main advantages of ddPCR over qPCR for cell-free DNA (cfDNA) applications?
51:27 Do we use qPCR thermocycler or need a separate thermocycler machine for running ddPCR?
53:51 How much does it cost compared to qPCR?
56:23 What is the benefit for cell and gene therapy applications of using Droplet Digital PCR technology vs qPCR technology?
59:57 Closing
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