JoVE Journal

Immunology and Infection

This content is Open Access.

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

13.3K Views

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10:00 min

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March 24th, 2015

DOI :

10.3791/52650-v

March 24th, 2015

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Lynnsey A. Renn¹, Terence C. Theisen¹, Maria B. Navarro¹, Viraj P. Mane¹, Lynnsie M. Schramm¹, Kevin D. Kirschman¹, Giulia Fabozzi¹, Philippa Hillyer¹, Montserrat Puig², Daniela Verthelyi², Ronald L. Rabin¹

¹Center for Biologics Evaluation and Research, US Food and Drug Administration, ²Center for Drug Evaluation and Research, US Food and Drug Administration

Summary

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Explore More Videos

Interferon Subtypes

Type I Interferons

Type III Interferons

Molecular Beacon Probes

Automated Pipetting

Gene Expression Profiling

Chapters in this video

0:05

Title

1:18

Preparation of Standard Serial Dilutions, Primer/Probe, and No Template Control Working Stock Mixes

3:18

Preparation of 384-well Assay Plates

5:47

Loading and Running the Assay Plate

8:13

Results: Analysis of Quantitative RT-PCR IFN Data

9:29

Conclusion

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