Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells: RT-PCR. Homo sapiens

Accurate profiling of RNA expression of single cells is a valuable approach for broadening our understanding of cancer biology and mechanisms of dissemination, but requires the development of reliable methods for their molecular characterization. Here we evaluate a single cell methodology which generates microgram amounts of cDNA suitable for next generation sequencing (RNA-Seq), high throughput RT-qPCR and Affymetrix array analysis. The approach was tested by comparing expression profiles of amplified single MCF7 and MCF10A cells to profiles generated from unamplified RNA. The expression profiles were compared by Affymetrix-U133 arrays, RNA-Seq and high-density qPCR. There were strong cross-platform correlations of >80% and concordance between single cell and unamplified material of >70%. We exemplify the approach through analysis of rare sorted cancer initiating cells (CICs) derived from a NSCLC patient-derived xenograft. Populations of 10 cells from total tumour and two distinct subsets of CIC, putatively involved in primary tumor maintenance or metastasis formation were FACS sorted then directly amplified. CIC expression profiles strongly correlated with published stem-cell and epithelial-mesenchymal transition (EMT) signatures. Our results confirm the utility of the amplification system and our methodology to detect and distinguish RNA profiles in rare cell populations that inform on EMT and stem-cell characteristics. This GEO dataset comprises the RT-qPCR data for MCF7 and MCF10A unamplified cDNA from 1μg RNA, RNA-AMP cDNA from 1ng RNA and RNA-AMP cDNA from single cell samples. Overall design: 22 samples. 3 biological replicates each for MCF7 cDNA generated from 1ng amplified RNA, MCF10A cDNA generated from 1ng amplified RNA, MCF7 cDNA generated from 1 μg unamplified RNA and MCF10A cDNA generated from 1 μg unamplified RNA. 5 biological replicates each for MCF7 cDNA generated from RNA amplified from a single cell and MCF10A cDNA generated from RNA amplified from a single cell. Expression of genes in MCF7 and MCF10A was compared in each of the sample types, and these results from each sample type were compared.

精准解析单细胞RNA表达谱，是加深我们对癌症生物学及肿瘤扩散机制理解的重要研究手段，但目前仍需开发可靠的分子表征方法。
本研究评估了一种单细胞实验方法，该方法可制备微克级互补脱氧核糖核酸（cDNA），适配下一代测序（RNA-Seq）、高通量实时定量逆转录PCR（RT-qPCR）以及Affymetrix芯片分析。
本研究通过比较扩增后的单细胞MCF7与MCF10A的表达谱与未扩增RNA所获得的表达谱，对该方法进行了验证。
研究采用Affymetrix U133芯片、RNA-Seq及高密度qPCR对表达谱进行了比对分析。
结果显示不同平台间相关性均高于80%，单细胞样品与未扩增样品的一致性亦高于70%。
我们通过分析非小细胞肺癌（NSCLC，non-small cell lung cancer）患者来源异种移植瘤中稀有分选得到的癌症起始细胞（CICs，cancer initiating cells），对该方法进行了实例验证。
我们从整体肿瘤组织中分离出10个细胞的群体，并分离出两种推测分别参与原发肿瘤维持与转移形成的CIC亚群，通过荧光激活细胞分选术（FACS，Fluorescence-Activated Cell Sorting）分选后直接进行扩增。
CIC的表达谱与已发表的干细胞特征及上皮间质转化（EMT，epithelial-mesenchymal transition）特征显著相关。
本研究结果证实了该扩增体系及实验方法的实用性，可用于检测并区分稀有细胞群体中反映EMT及干细胞特征的RNA表达谱。
本基因表达综合数据库（GEO，Gene Expression Omnibus）数据集包含如下RT-qPCR数据：源自1μg RNA的MCF7与MCF10A未扩增cDNA、源自1ng RNA的RNA-AMP cDNA，以及单细胞样品的RNA-AMP cDNA。
实验整体设计：共包含22个样本。其中，源自1ng扩增RNA的MCF7 cDNA、源自1ng扩增RNA的MCF10A cDNA、源自1μg未扩增RNA的MCF7 cDNA，以及源自1μg未扩增RNA的MCF10A cDNA各设3个生物学重复；单细胞RNA扩增得到的MCF7 cDNA与MCF10A cDNA各设5个生物学重复。本研究对各类样本中MCF7与MCF10A的基因表达进行了比较，并对不同样本类型的实验结果进行了交叉比对。