Evaluation and validation of a robust single cell RNA-amplification protocol through transcriptional profiling of enriched lung cancer initiating cells

BMC Genomics

Ged Brady⁶  Caroline Dive⁶  Luca Roz⁵  Giulia Bertolini⁵  Fiona Blackhall¹  Crispin Miller³  Stuart Pepper²  Massimo Moro⁵  Suzanne Faulkner⁶  Louise Carter⁶  Yvonne Hey²  Gillian Newton²  Catriona Tate⁶  Mahmood Ayub⁶  Yaoyong Li⁴  Dominic G Rothwell⁶ 

[1] Christie NHS Foundation Trust, Institute of Cancer Sciences, University of Manchester, Manchester M20 4BX, UK;Molecular Biology Core Facility, CR-UK Manchester Institute, University of Manchester, Manchester M20 4BX, UK;RNA Biology Group, CR-UK Manchester Institute, University of Manchester, Manchester M20 4BX, UK;Computational Biology Support, CR-UK Manchester Institute, University of Manchester, Manchester M20 4BX, UK;Department of Experimental Oncology, Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano 20133, Italy;Nucleic Acid Biomarker Laboratory, Clinical & Experimental Pharmacology, CR-UK Manchester Institute, University of Manchester, Manchester M20 4BX, UK

关键词: Cancer initiating cell;    Microarray;    RNA-Seq;    Transcriptional profiling;    Single cell;    RNA-Amplification;   

Others  :  1127142
DOI  :  10.1186/1471-2164-15-1129

 received in 2014-09-09, accepted in 2014-12-11,  发布年份 2014

【 摘 要 】

Background

Although profiling of RNA in single cells has broadened our understanding of development, cancer biology and mechanisms of disease dissemination, it requires the development of reliable and flexible methods. Here we demonstrate that the EpiStem RNA-Amp™ methodology reproducibly generates microgram amounts of cDNA suitable for RNA-Seq, RT-qPCR arrays and Microarray analysis.

Results

Initial experiments compared amplified cDNA generated by three commercial RNA-Amplification protocols (Miltenyi μMACS™ SuperAmp™, NuGEN Ovation® One-Direct System and EpiStem RNA-Amp™) applied to single cell equivalent levels of RNA (25–50 pg) using Affymetrix arrays. The EpiStem RNA-Amp™ kit exhibited the highest sensitivity and was therefore chosen for further testing. A comparison of Affymetrix array data from RNA-Amp™ cDNA generated from single MCF7 and MCF10A cells to reference controls of unamplified cDNA revealed a high degree of concordance. To assess the flexibility of the amplification system single cell RNA-Amp™ cDNA was also analysed using RNA-Seq and high-density qPCR, and showed strong cross-platform correlations. To exemplify the approach we used the system to analyse RNA profiles of small populations of rare cancer initiating cells (CICs) derived from a NSCLC patient-derived xenograft. RNA-Seq analysis was able to identify transcriptional differences in distinct subsets of CIC, with one group potentially enriched for metastasis formation. Pathway analysis revealed that the distinct transcriptional signatures demonstrated in the CIC subpopulations were significantly correlated with published stem-cell and epithelial-mesenchymal transition signatures.

Conclusions

The combined results confirm the sensitivity and flexibility of the RNA-Amp™ method and demonstrate the suitability of the approach for identifying clinically relevant signatures in rare, biologically important cell populations.

【 授权许可】

   
2014 Rothwell et al.; licensee BioMed Central.

【 图 表 】

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