High-Throughput Single-Cell Real-Time Quantitative PCR Analysis
Examining transcriptomics of populations at the single-cell level allows for higher resolution when studying functionality in development, differentiation, and physiology. Real-time quantitative PCR (qPCR) enables a sensitive detection of specific gene ex
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Introduction Differences in gene expression determine the development, differentiation, and physiology of an organism. The most common methods to explore gene expression average the expression profiles over large numbers of cells. This obscures population heterogeneity and whether there are one or more distinct cell types within a population [1–3]. Quantitative PCR (qPCR), also known as real-time PCR, is one method to determine transcript or DNA region levels in a sample. Gene-specific assays are used to amplify the corresponding gene using PCR, and additional fluorescence probe for monitoring the amplification. The major difficulty when applying qPCR to singlecell study is the enormous number of reactions required and the reagent cost for those experiments, due to the need of increased sample number. The use of the nanofluidic system offers the option to automatically combine up to 96 assays (measured genes) with 96 samples and detect the amplification process of all the combined reactions, which allows for higher throughput of cell number. The systems uses nanofluidic chips which results in a much lower costs for reagents.
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_11, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Liora Haim-Vilmovsky
Further advantages of this method include reproducibility, sensitivity, and specificity [4–6]; however, it is still limited to less than a hundred assays/samples per chip, and lower accuracy when measuring low expressed transcripts [7]. Other methods are available today to detect the general transcriptomics of a large number of cells with lower costs. However, this technique can be used to validate the findings and can be used in case there is urgency in the required results and its analysis.
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Materials Work in an RNAse, DNase, DNA, and PCR products-free laminar flow hood.
2.1
FACS
1. 96-Well PCR plates (see Note 1). 2. RNase inhibitor. Store at
20 C.
3. CellDirect 2 reaction Mix (Invitrogen). Store at
20 C.
4. Lysis buffer: CellDirect 2 reaction Mix, 2% RNase inhibitor. Store at 20 C. 5. FACS instrument. 6. Adhesive plate seals. 7. Dry ice. 2.2 Reverse Transcription and Specific Target Amplification
1. 20 TaqMan Gene Expression Assays (see Notes 2 and 3). Store at 20 C. 2. DNA suspension Buffer: 10 mM Tris, pH 8.0, 0.1 mM EDTA. Store at 4 C. 3. 0.2 Assay mix: Each assay in final concentration of 0.2, in DNA suspension Buffer (see Note 4). Store at 20 C. 4. SuperScript III RT/Platinum Taq Mix. Store at
20 C.
5. PCR certified water. Store at room temperature. 6. 96-Well thermal cycler. 2.3
Real-Time PCR
1. 2 Assay Loading Reagent (Fluidigm) (see Note 5). Store at 20 C. 2. Quanta PerfeCTa qPCR Fast Mix, low ROX. Store at
20 C.
3. 20 GE Sample Loading Reagent (Fluidigm) (see Note 5). Store at 20 C. 4. 96.96 dynamic array (Chip). 5. Integrated Fluidic Circuit (IFC) Controller HX. 6. Biomark
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