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Insight & Intelligence™: Jul 23, 2014

Sorting FISH

This literature review discusses how researchers used FISH to detect RNA transcripts at the single-cell level.

Sorting FISH

Flow cytometry can be applied to RNA detection enabling cell sorting based on the RNA transcripts of interests. [© KaYann - Fotolia.com]

    Figure. Single-cell GFP RNA and protein quantification. (a) GFP induction quantified by labeled mRNA fluorescence (upper), RT-qPCR normalized to Oct4 (middle), and protein fluorescence (lower). (b) GFP induced (1 µg/mL doxycycline) and uninduced mESC mixture with indicated positive (red) and negative (black) sorting gates. (c) RT-qPCR (normalized to Oct4) for GFP mRNA in each sorted fraction. (d) Single cell correlation of GFP mRNA and protein fluorescence in iPSCs heterozygous for Oct4-IRES-GFP (iPS-2i; see the article's Supplementary Information at http://dx.doi.org/10.1038/nmeth.2910). (e) Correlation and linear scaling of GFP protein with mRNA in single cells for representative doxycycline induction levels. GFP, green fluorescent protein; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; mESCs, mouse embryonic stem cells; iPSCs, induced pluripotent stem cells.
    Click Image To Enlarge +
    Figure. Single-cell GFP RNA and protein quantification. (a) GFP induction quantified by labeled mRNA fluorescence (upper), RT-qPCR normalized to Oct4 (middle), and protein fluorescence (lower). (b) GFP induced (1 µg/mL doxycycline) and uninduced mESC mixture with indicated positive (red) and negative (black) sorting gates. (c) RT-qPCR (normalized to Oct4) for GFP mRNA in each sorted fraction. (d) Single cell correlation of GFP mRNA and protein fluorescence in iPSCs heterozygous for Oct4-IRES-GFP (iPS-2i; see the article's Supplementary Information at http://dx.doi.org/10.1038/nmeth.2910). (e) Correlation and linear scaling of GFP protein with mRNA in single cells for representative doxycycline induction levels. GFP, green fluorescent protein; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; mESCs, mouse embryonic stem cells; iPSCs, induced pluripotent stem cells.

    Fluorescent in situ hybridization (FISH) is a sensitive method to detect specific sequences in either DNA or RNA. This article demonstrates the application of FISH to detect RNA transcripts at the single-cell level. Degradation of the mRNA during extraction from cells or tissues is the primary factor that has limited the application of FISH. The authors* addressed this issue by developing an RNA-preserving hybridization buffer that maintains the RNA integrity following hybridization and coupling the analysis to flow cytometry.

    A proof-of-principle experiment is shown in which both high and low green fluorescent protein (GFP)-expressing cells were sorted using FACS, and it was demonstrated that both GFP fluorescence and mRNA fluorescence was concordant (see Figure). The resolution of the method was improved by measuring RNA abundance in particular cell-cycle phases—with the use of a 30-nt FISH probe library, 13 RNA molecules could be detected. The method was then applied to measure transcripts during iPSC reprogramming.

    Cells expressing the reprogramming transcription factor Sox2 were profiled in a background of cells that did not express Sox2, and these results were compared to the profile of iPSCs. This showed a subpopulation of cells in the Sox2-expressing cells that closely matched the iPSCs. This method allows flow cytometry to be applied to RNA detection enabling sorting of cells based on the RNA transcripts of interests.

    *Abstract from Nature Methods 2014, Vol. 11: 549–551

    We have developed a quantitative technique for sorting cells on the basis of endogenous RNA abundance, with a molecular resolution of 10–20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high-fidelity transcriptome measurement of mouse induced pluripotent stem cells (iPSCs) isolated from a heterogeneous reprogramming culture. This method is broadly applicable to profiling transcriptionally distinct cellular states without requiring antibodies or transgenic fluorescent proteins.

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