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Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts
JoVE Journal
Genetics
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JoVE Journal Genetics
Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

DOI:
10.3791/54473-v

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11:19 min

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October 09, 2016

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, , ,
1Department of Biomolecular Chemistry,University of Wisconsin-Madison, 2Department of Medicine,University of Wisconsin-Madison

Chapters

  • 00:05Title
  • 01:16Creating Plasmids as Template Standards
  • 02:20Analyzing Primer Specificity for Absolute qPCR
  • 05:49Performing Relative qPCR Assays
  • 06:57Analyzing Absolute qPCR Data for Unknown Samples
  • 08:27Results: Measurement of STAT3 Splice Variant Levels in Eosinophils Treated with Cytokines
  • 10:06Conclusion

Summary

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Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and α/β C-termini, can be quantified.

Tags

Keywords: Quantitative PCRSTAT3 Splice VariantsEosinophilsAbsolute QuantificationRelative QuantificationPlasmid StandardsCytokine StimulationCDNASequencingPlasmid ConcentrationCopy NumberDilution SeriesNon-target ControlsTarget MixPrimer Pairs

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