细胞群体和组织表现出独特的基因表达谱,允许表征和区分细胞亚型。监测细胞类型特异性标志物的基因表达可以指示细胞状态,如增殖,压力,静止,或成熟。定量逆转录酶PCR(qRT-PCR)允许定量细胞类型特异性标志物的RNA表达并区分一种细胞类型与另一种细胞类型。然而,qRT-PCR方法(例如TaqMan技术)需要荧光报告基因来表征靶基因,并且难以扩大规模,因为它们需要不同的探针用于每个反应。大容量或单细胞RNA转录组学是耗时且昂贵的。处理RNA测序数据可能需要数周时间,这对于质量控制和监测基因表达不是最佳的,例如,在诱导多能干细胞(iPSC)分化为特化细胞类型的范例中。一种更具成本效益的测定法基于SYBRGreen技术。SYBRGreen是一种与双链DNA结合的核酸染料,吸收497nm的蓝光,并在插入双链DNA后在520nm处发出高达1,000倍的绿光。当相对于管家基因进行归一化并与对照条件进行比较时,可以基于荧光强度水平来定量感兴趣区域的扩增。以前,我们建立了SYBRGreenqRT-PCR方案,以使用涂布在96孔板上的一组有限的标记物表征样品。这里,我们优化了这个过程,增加了一个384孔格式的通量,并比较mRNA表达,以区分iPSC衍生的神经元亚型彼此通过增加基因的数量,细胞类型,和分化时间点。在这个协议中,我们开发了以下内容:i)使用Primer3软件的命令行版本,我们为感兴趣的基因更容易和快速地设计引物;ii)使用384孔板格式,电子多通道移液器,和移液机器人,我们在单个平板上分析了四倍多的基因,同时使用与96孔板相同体积的试剂。该方案的优点是增加了SYBRGreen测定的吞吐量,同时限制了移液错误/不一致,试剂使用,成本,和时间。图形概述。
Cell populations and tissues exhibit unique gene expression profiles, which allow for characterizing and distinguishing cellular subtypes. Monitoring gene expression of cell type-specific markers can indicate cell status such as proliferation, stress, quiescence, or maturation. Quantitative reverse transcriptase PCR (qRT-PCR) allows quantifying RNA expression of cell type-specific markers and distinguishing one cell type from another. However, qRT-PCR methods such as TaqMan technology require fluorescent reporters to characterize target genes and are challenging to scale up as they need different probes for each reaction. Bulk or single-cell RNA transcriptomics is time-consuming and expensive. Processing RNA sequencing data can take several weeks, which is not optimal for quality control and monitoring gene expression, e.g., during a differentiation paradigm of induced pluripotent stem cells (iPSCs) into a specialized cell type. A more cost-effective assay is based on SYBR Green technology. SYBR Green is a nucleic acid dye that binds to double-stranded DNA, absorbs blue light at 497 nm, and emits green light at 520 nm up to 1,000-fold upon intercalation with double-stranded DNA. Amplification of a region of interest can be quantified based on the level of fluorescence intensity when normalized to a housekeeping gene and compared to control conditions. Previously, we established a SYBR Green qRT-PCR protocol to characterize samples using a limited set of markers plated on a 96-well plate. Here, we optimize the process and increase throughput to a 384-well format and compare mRNA expression to distinguish iPSC-derived neuronal subtypes from each other by increasing the number of genes, cell types, and differentiation time points. In this protocol, we develop the following: i) using the command-line version of Primer3 software, we design primers more easily and quickly for the gene of interest; ii) using a 384-well plate format, electronic multichannel pipettes, and pipetting robots, we analyze four times more genes on a single plate while using the same volume of reagents as in a 96-well plate. The advantages of this protocol are the increased throughput of this SYBR Green assay while limiting pipetting errors/inconsistencies, reagent use, cost, and time. Graphical overview.