WEBINAR - Expert Coffee Chats - Real-Time PCR in Infectious Disease

Join us for a chat with Bio-Rad application scientists about the current issues most relevant to your research. Jump to specific topics by following the links below.
In this episode, we discuss: Real-Time PCR in Infectious Disease
- Characterizing COVID-19 using qPCR
- Accurately select your internal control
- Exploring 1-step vs 2-step in sample preparation
00:10 Introduction
3:49 What is qPCR, and why is it useful in the detection and research of infectious disease?
5:23 What makes for a good qPCR assay?
7:50 What is the smallest amount that can qPCR detect?
9:18 What are the conditions that I need to consider while designing PCR primers?
13:33 If I have 4 samples; all are reverse transcribed with 1µg of RNA into cDNA; I take 100 ng of each cDNA and run a housekeeping gene (like ACTB or TBP) using FAM probes. Why are the Ct values for all samples not exactly the same-since the starting input was the same?
16:25 How does qPCR compare against ddPCR in terms of utility in pathogen detection applications?
19:48 If you run an RT-PCR for 50 cycles for example would you accept as positive a sample with CT value of 49? How would you suggest to set thresholds and baselines?
25:10 I have seen labs that create standard curves with their samples; for 4 samples, they take 5 µl of each sample, mix it and run a housekeeping gene on it with serial dilutions to create a standard curve. Then they read samples off this curve. When they have more samples (from the same study), they create another standard curve from the new samples. I don't think this is correct; I would like to have your thoughts on this too. Thank you!
28:12 What are some of the most common applications of qPCR for pathogen testing in food safety, water quality, and environmental areas?
32:24 How frequently do I need to calibrate fluoros or thermo for my instrument maintenance except it goes really off? [How do I need to account for thermo-validation and fluorophore validation within a thermocycler?]
34:53 Is it always best practice to let the PCR machine set the threshold/baseline? The CFX does this and I like to trust it!
38:38 What are the new instruments coming in qPCR?
40:53 What is the purpose of the calibrator sample when running a qPCR?
43:04 Why is qPCR less widely used when testing targets in cell-free DNA e.g. rare somatic mutation detection?
46:55 I used one 384-well plate in Quant Studio 5 and the other in Applied Biosystems machine, at the same time, using SYBR® Green. Can I use the data from two machines and their CTs into one plot to comprehend the data? Please help..!
48:39 I've heard hydrolysis probes are more specific, should I be using those instead of SYBR Green?
51:16 With the increased demand for qPCR supplies, what can I do to increase my throughput and use fewer reagents? Are there any downsides or caveats to doing this?
53:02 How do I ensure that my qPCR follows literature and industry scientific rigor?
57:32 Closing
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