Highly Parallel Genome-wide Expression Analysis of Single Mammalian Cells

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2012 Feb 21

PMID 22347404

Citations 19

Authors

Jian-Bing Fan

Jing Chen

Craig S April

Jeffrey S Fisher

Brandy Klotzle

Marina Bibikova

Fiona Kaper

Mostafa Ronaghi

Sten Linnarsson

Takayo Ota

Jeremy Chien

Louise C Laurent

Jeanne F Loring

Sean V Nisperos

Gina Y Chen

Jiang F Zhong

Affiliations

Soon will be listed here.

Abstract

Background: We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.

Methodology/principal Findings: The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.

Conclusions/significance: In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology.

Citing Articles

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.

Manasa Bhamidimarri P, Salameh L, Mahdami A, Abdullah H, Mahboub B, Hamoudi R Heliyon. 2024; 10(12):e32896.

PMID: 38988576 PMC: 11234047. DOI: 10.1016/j.heliyon.2024.e32896.

CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.

Oguri Y, Shinoda K, Kim H, Alba D, Bolus W, Wang Q Cell. 2020; 182(3):563-577.e20.

PMID: 32615086 PMC: 7415677. DOI: 10.1016/j.cell.2020.06.021.

Improving single-cell transcriptome sequencing efficiency with a microfluidic phase-switch device.

Zhang B, Xu H, Huang Y, Shu W, Feng H, Cai J Analyst. 2019; 144(24):7185-7191.

PMID: 31688860 PMC: 6925944. DOI: 10.1039/c9an00823c.

Single-cell transcriptomes reveal the mechanism for a breast cancer prognostic gene panel.

Li S, Stucky A, Chen X, Kabeer M, Loudon W, Plant A Oncotarget. 2018; 9(70):33290-33301.

PMID: 30279960 PMC: 6161791. DOI: 10.18632/oncotarget.26044.

Relapse pathway of glioblastoma revealed by single-cell molecular analysis.

Chen X, Wen Q, Stucky A, Zeng Y, Gao S, Loudon W Carcinogenesis. 2018; 39(7):931-936.

PMID: 29718126 PMC: 6248540. DOI: 10.1093/carcin/bgy052.

References

Spiess A, Mueller N, Ivell R . Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations. BMC Genomics. 2003; 4(1):44. PMC: 280674. DOI: 10.1186/1471-2164-4-44. View

Zhang S, Balch C, Chan M, Lai H, Matei D, Schilder J . Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res. 2008; 68(11):4311-20. PMC: 2553722. DOI: 10.1158/0008-5472.CAN-08-0364. View

Singh R, Maganti R, Jabba S, Wang M, Deng G, Heath J . Microarray-based comparison of three amplification methods for nanogram amounts of total RNA. Am J Physiol Cell Physiol. 2004; 288(5):C1179-89. PMC: 2020524. DOI: 10.1152/ajpcell.00258.2004. View

Kim T, Sul J, Peternko N, Lee J, Lee M, Patel V . Transcriptome transfer provides a model for understanding the phenotype of cardiomyocytes. Proc Natl Acad Sci U S A. 2011; 108(29):11918-23. PMC: 3141937. DOI: 10.1073/pnas.1101223108. View

Appay V, Bosio A, Lokan S, Wiencek Y, Biervert C, Kusters D . Sensitive gene expression profiling of human T cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages. J Immunol. 2007; 179(11):7406-14. DOI: 10.4049/jimmunol.179.11.7406. View

Kapteyn J, He R, McDowell E, Gang D . Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples. BMC Genomics. 2010; 11:413. PMC: 2996941. DOI: 10.1186/1471-2164-11-413. View

Froussard P . rPCR: a powerful tool for random amplification of whole RNA sequences. PCR Methods Appl. 1993; 2(3):185-90. DOI: 10.1101/gr.2.3.185. View

Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N . mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 2009; 6(5):377-82. DOI: 10.1038/nmeth.1315. View

Ramos C, Bowman T, Boles N, Merchant A, Zheng Y, Parra I . Evidence for diversity in transcriptional profiles of single hematopoietic stem cells. PLoS Genet. 2006; 2(9):e159. PMC: 1584276. DOI: 10.1371/journal.pgen.0020159. View

10.

Kiguchi K, Iwamori M, Mochizuki Y, Kishikawa T, Tsukazaki K, SAGA M . Selection of human ovarian carcinoma cells with high dissemination potential by repeated passage of the cells in vivo into nude mice, and involvement of Le(x)-determinant in the dissemination potential. Jpn J Cancer Res. 1998; 89(9):923-32. PMC: 5921947. DOI: 10.1111/j.1349-7006.1998.tb00650.x. View

11.

Bahar R, Hartmann C, Rodriguez K, Denny A, Busuttil R, Dolle M . Increased cell-to-cell variation in gene expression in ageing mouse heart. Nature. 2006; 441(7096):1011-4. DOI: 10.1038/nature04844. View

12.

Talasaz A, Powell A, Huber D, Berbee J, Roh K, Yu W . Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc Natl Acad Sci U S A. 2009; 106(10):3970-5. PMC: 2645911. DOI: 10.1073/pnas.0813188106. View

13.

Koirala S, Corfas G . Identification of novel glial genes by single-cell transcriptional profiling of Bergmann glial cells from mouse cerebellum. PLoS One. 2010; 5(2):e9198. PMC: 2820553. DOI: 10.1371/journal.pone.0009198. View

14.

Morris J, Singh J, Eberwine J . Transcriptome analysis of single cells. J Vis Exp. 2011; (50). PMC: 3376915. DOI: 10.3791/2634. View

15.

Petalidis L, Bhattacharyya S, Morris G, Collins V, Freeman T, Lyons P . Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis. Nucleic Acids Res. 2003; 31(22):e142. PMC: 275579. DOI: 10.1093/nar/gng142. View

16.

Dafforn A, Chen P, Deng G, Herrler M, Iglehart D, Koritala S . Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis. Biotechniques. 2004; 37(5):854-7. DOI: 10.2144/04375PF01. View

17.

Islam S, Kjallquist U, Moliner A, Zajac P, Fan J, Lonnerberg P . Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 2011; 21(7):1160-7. PMC: 3129258. DOI: 10.1101/gr.110882.110. View

18.

Morse A, Carballo V, Baldwin D, Taylor C, McIntyre L . Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems. J Biomol Tech. 2010; 21(3):141-7. PMC: 2922837. View

19.

Esumi S, Wu S, Yanagawa Y, Obata K, Sugimoto Y, Tamamaki N . Method for single-cell microarray analysis and application to gene-expression profiling of GABAergic neuron progenitors. Neurosci Res. 2008; 60(4):439-51. DOI: 10.1016/j.neures.2007.12.011. View

20.

Irimia D, Mindrinos M, Russom A, Xiao W, Wilhelmy J, Wang S . Genome-wide transcriptome analysis of 150 cell samples. Integr Biol (Camb). 2009; 1(1):99-107. PMC: 3763906. DOI: 10.1039/b814329c. View