Abstract
BackgroundAlthough 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.ResultsInitial 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1129) contains supplementary material, which is available to authorized users.
Highlights
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
Single cell equivalent amounts (25-50 pg) of pooled RNA isolated from the human epithelial cell lines MCF7 were amplified in duplicate and 5 μg of cDNA from each sample run on an Affymetrix array
Column headings are: metastatic-associated cancer initiating cells (MCIC) - metastasis associated cancer initiating cells; RCIC- resident cancer initiating cells; TT - unfractionated total tumour (TT); Taube et al - epithelial to mesenchymal transition (EMT) genes identified by Taube and colleagues [27]; Loboda et al – EMT genes identified by Loboda and colleagues [21]; Blick et al - EMT genes identified by Blick and colleagues [22]
Summary
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. Single cell studies utilized cDNA microarrays [8] which enable quantification of tens of thousands of known genes [9,10] This technology has limitations including a restricted fold-range of detection and potential cross-hybridisation between similar sequences [11], as well as being restricted to the probe sets present on the array. A third platform that has been used to analyse transcriptional signatures of single cells is high-density qPCR, which provides a more restricted but targeted approach with a wide dynamic range and can be readily transferred to a clinical setting [14].
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