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Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards
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Genetics
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JoVE Journal Genetics
Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

DOI:
10.3791/55219-v

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10:50 min

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February 25, 2017

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, ,
1Laboratory of Pathology, Center for Cancer Research,National Cancer Institute, 2Experimental Transplantation and Immunology Branch, Center for Cancer Research,National Cancer Institute

Chapters

  • 00:05Title
  • 00:56Preparation of Standards from Purified DNA Amplicons and Treatment of MCF7 p53-Venus Cells
  • 01:53Preparation of Lysis Buffer for Cell Sorting
  • 04:15Cell Sorter Setup, Cell Harvesting, and Sorting
  • 05:47Exonuclease Treatment and Sort Quality Control Using qPCR
  • 07:02Gene Expression Measurement Using Microfluidic qPCR
  • 09:03Results: Gene Expression Profiling in Single Cells Using FACS Analysis and qPCR
  • 10:09Conclusion

Summary

Automatic Translation

Automatic Translation

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Tags

Single-cell Gene ExpressionFACSQPCRInternal StandardsMicrofluidicTranscript QuantificationRNA-SeqCell SortingAmpliconNeocarzinostatinLysis BufferE. Coli DNAStandards

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