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Single-cell Gene Expression Profiling of Mouse Stem Cells With Fluidigm BiomarkTM Dynamic Array
采用Fluidigm Biomark™动态芯片检测小鼠干细胞中的单细胞基因表达谱   

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Abstract

This protocol describes how to use Fluidigm BiomarkTM 96.96 dynamic arrays for high-throughput expression profiling from single mouse stem cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment. This Dynamic Array contains a network of microfluidic channels, chambers and valves that automatically assemble all these individual PCR reactions. Single-cell profiling can provide definitive evidence of stem cell heterogeneity. Modifications are most likely needed if users intend to use BiomarkTM 48.48 Dynamic array or experimenter-designed primers in conjunction with DNA-binding dyes such as EvaGreen (Biotium 31000).

Figure 1. Workflow for Single-Cell Gene Expression Profiling in Mouse Stem Cells using Fluidigm BiomarkTM Dynamic  Array. The main basic steps of this protocol are: Single cell sorting on 96 well plates, specific target amplifycation, chip loading, single cell gene expression RT-PCR and data collection. This Fluidigm Dynamic Array integrated fluidic circuit chip (IFCs) contains a network of microfluidic channels, chambers and valves that automatically assemble individual PCR reactions. More specific information about this particular chip can be obtained from http://www.fluidigm.com/single-cell-gene-expression.html.

Materials and Reagents

  1. Mouse ES cells (E14 Cell Line) (ATCC®, catalog number: SCRC-1040TM )
  2. Low-EDTA (0.1 mM EDTA) TE buffer (Teknova, catalog number: T0227 )
  3. CellsDirectTM One-Step qRT-PCR kit (Life Technologies, InvitrogenTM, catalog number: 11753-100 )
  4. Taqman Gene Expression Assays of the genes of interest for your study (Applied Biosystems)
    Note: TaqMan® Assays are probe and primer sets based on 5’ nuclease chemistry using TaqMan® MGB (minor groove binder) probes.Search your Taqman gene expression assays here: https://bioinfo.appliedbiosystems.com/genome-database/gene-expression.html
    1. Select the type of experiment you are conducting. Click In gene expression
    2. Select the type of assay you want. click in all gene expression assays
    3. Select what specie you want to target. Click on mouse since our samples are Mouse ES cells.
    4. Enter the target information. Add the gene symbol or entrez gene symbol or entrez gene  ID of the gene you want to analyze its expression.
  5. 96.96 Dynamic Array Chip for Gene Expression (Fluidigm, catalog number: BMK-M-96.96 )
  6. Gene expression 96.96 Dynamic Array sample & loading reagent kit (Fluidigm, catalog number: 85000802 ), which contains:
    1. Two tubes of 2x assay loading reagent (Fluidigm, catalog number: 85000736 )
    2. Two tubes of 20x GE sample loading reagent (Fluidigm, catalog number: 85000746 )
    3. 20 syringes of Control Line Fluid (150 μl)
  1. TaqMan® universal PCR master mix (Applied Biosystems, catalog number: 4304437 )
  2. Dulbecco's Phosphate-Buffered Saline (DPBS) (Life Technologies, catalog number: 14190-136 )
  3. TryPLE express solution (Life Technologies, catalog number: 12605-010 )
  4. 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies, catalog number: D1306 )
  5. Propidium iodide (PI) (Life Technologies, catalog number: P-3566 )
  6. 2x Assay Loading Reagent (Fluidigm, catalog number: 85000736)
  7. 2x TaqMan®Universal PCR Master Mix (Applied Biosystems, catalog number: 4304437)
  8. 20x GE Sample Loading Reagent (Fluidigm, catalog number: 85000746 )

Equipment

  1. 96-well semi-skirted plate, 0.2 ml wells, straight raised sides (USA scientific, catalog number: 1402-9200 )
  2. Optical adhesive film (Applied Biosystems, catalog number: 4311971 )
  3. PCR-8-Tube Strips & Strip Camps, natural (USA Scientific, catalog number: 1402-2500 )
  4. TipOne 0.1–10 μl extended-length filter tips (USA Scientific, catalog number: 1120-3810 )
  5. TipOne 101–1,000 μl extended-length filter tips (USA Scientific, catalog number: 1122-1830 )
  6. TipOne 1–200 μl graduated filter tips (USA Scientific, catalog number: 1120-8810 )
  7. Fluorescence-activated cell sorter (FACS Aria II; BD Biosciences, catalog number: 643756 )
  8. Multichannel pipettes (10-100 μl Eppendorf Research®, catalog number: 38-3122000043 ; 0.5-10 μl catalog number: 38-3122000019 )
  9. Thermal cycler (VeritiTM 96-well Applied Biosystems, catalog number: PN 4375786 )
  10. The Integrated Fluidic Circuit IFC-Controllers
  11. The MX model primes and loads 48.48 chips
  12. The HX model primes and loads the 96.96 chips.
  13. Centrifuge (Eppendorf, catalog number: 5810 )

Procedure

  1. Reverse transcription-specific target amplification (RT-STA) master mix preparation.
    1. Thaw the CellsDirect 2x Reaction Mix from the CellsDirectTM One-Step qRT-PCR kit. All steps should be performed on ice, and reagents should be chilled and/or thawed immediately prior to use.
    2. Transfer 65 μl of the 2x Reaction Mix to each tube of the PCR-8-Tube Strip.
    3. Add 5 μl of 2x Reaction Mix from the PCR-8-Tube Strip to each well of the 96 well-plate with the multichannel pipette.
      Since the amount of RNA from a single cell is few pictograms, we don’t perform a traditional RNA isolation protocol. Instead, the sorted cells are directly introduced into the 2x Reaction Mix. To ensure optimal sample preservation, this master mix contains CellDirect 2x reaction Mix (CellsDirect One-Step qRT-PCR kit) and SUPERase-In. This enzyme protects the RNA extracted from a lysed cell from any RNAases that could be present in the RT-STA master mix.

  2. Cell sorting
    1. Detach the mouse ES cells with TryPLE Express solution. Gently aspirate away cell media culture and wash the cells with 5-6 ml of fresh 1x DPBS to remove serum that could inhibit TryPLE Express solution activity.
    2. Aspirate away the 1x DPBS.
    3. Add 1 ml of pre-warmed TryPLE Express solution directly to the cell culture plate, and incubate the plate at 37 °C, 5% CO2 for 3-5 min.
    4. Take the cell culture plate out, gently tap or shake it, and then add 10 ml of fresh cell culture medium with 10% (v/v) FBS to stop trypsin activity. Mix cells with a pipette and transfer 1 x 106 cells into a 15 ml conical tube. Centrifuge at 300 x g, 4 °C for 5 min.
    5. Aspirate the cell supernatant and resuspend 1 x 106 cells in 1 ml of 1x DPBS supplemented with 5% (v/v) FBS.
    6. Keep 1 x 106 cells on ice before sorting or label the cells with the suitable surface markers according to the cell population you are interested to study.
    7. Add 10 μl of DAPI (> 200 ng/ml) or 10 μl of PI (5 μg/ml) per 1 x 106 cells to the single-cell suspension that contains 1 x 106 cells suspended in 1 ml of 1x DPBS supplemented with 5% (v/v) FBS just before cell sorting to distinguish the live cell population.
    8. Perform cell sorting in FACS Aria II and data analysis with FACS Diva software.
    9. When sorting cells gate for singlet’s in your sorting parameters.
    10. Sort single cells directly into the wells of a 96 well-plate containing a 5 μl mixture of CellsDirect 2x reaction mix (component CellsDirect One-Step qRT PCR Kits, Invitrogen). We estimate that the final volume of the reaction after cells are sorted into the wells will be 6 μl.
      Note: Sort the cells into the same plate that is used for the RT-STA PCR. Always keep the well-plate on ice.
    11. Centrifuge at 300 x g, 4 °C for 5 min the 96 well-plate to let the cells go into the CellsDirect 2x reaction mix.
    12. Seal the plate with the optical adhesive film.
    13. Store the plate at -80 °C. This overnight freezing step allows a better lysis efficiency.

  3. Preparation of the Specific Target Amplification (RT- STA) master mix.
    1. Make an excel file template for the 96 well-plate and label each cell with the specific 20x Taqman assay probe.
    2. Thaw the 20x Taqman assays and deposit them in the 96 well-plate according to the location establish in the excel file template for each 20x Taqman assay probe.
    3. Thaw the plate with the sorted cells in the CellsDirect 2x reaction mix on ice.
    4. Prepare the assay mix in a 1.5 ml eppendorf tube. Pool all Taqman real-time assays that will be analyzed in the single-cell real-time PCR that you previously deposited on a 96 well-plate in step b. Final concentrations of the Taqman assays used in the mix will be 0.2x. Use TE buffer to dilute the Taqman assays. This is the 0.2x assay mix.
      Component
      Volume to add per reaction (μl)
      Final concentration
      20x Taqman gene expression assay1
      1.5
      0.2x
      20x Taqman gene expression assay2
      1.5
      0.2x



      TE buffer
      Up to 150 μl

      TOTAL
      150 μl

    5. Prepare the sample RT-STA Master Mix by combining the following reagents:
      Component
      Volume (μl)
      0.2x Assay Mix
      2.5
      SuperScriptTM RT/Platinum® Taq Mix
      0.2
      TE Buffer
      1.3
      TOTAL
      4
      Note: It is important to create a bulk solution for all the samples being tested to overcome pipetting limitations.
    6. Add 4 μl of the RT-STA master mix to each well that already contain the sorted cell immersed into the CellsDirect 2x reaction mix.
    7. Seal the plate with the optical adhesive film. Leave the plate on ice for 5 min meantime you program the thermal cycler.
      Before you run the Reverse transcription and specific target amplification verify that each well from the 96 well plate has the components and the volumes according to the table below. Your final volume has to be 9 μl.
      Component Volume (μl)
      CellsDirect 2x Reaction Mix (This reagent is already in the plate with the sorted cell)
      5.0
      0.2x Assay Mix
      2.5
      SuperScriptTM RT/Platinum® Taq Mix
      0.2
      TE Buffer
      1.3
      TOTAL
      9

  1. Reverse transcription and specific target amplification.
    1. Place the 96 well plate with the RT-STA master mix in a thermal cycler.
      50 °C for 15 min (Step to reverse transcribe the RNA to cDNA)
      95 °C for 2 min (Step to inactivate the RT enzyme and activate the Taq)
      22 Cycles of 95 °C for 15 sec and 60 °C for 4 min (STA Specific Target Amplification)
      Hold at 4 °C
    2. Run the sample PCR according to these parameters:
      Note: We screened different number of cycles and we found that for single cell analysis 22 cycles gave us better results in the Chip resolution.
    3. Dilute the resulting cDNA product 1:5 with TE buffer.
      Any dilution between 1:2 and 1:5 is possible.
  1. Preparing 10x Assays
    In a DNA-free hood, prepare a 96 well-plate with 5μl aliquots of 10x assays using the volumes in the table below (scale up appropriately for multiple runs). Add the 20x TaqMan assays, one per well, to the 96 well-plate in the same position as in the excel file template.
    Component
    Volume per Inlet (μl)
    20x Taqman® gene expression assay (Applied Biosystems)
    2.5
    2x Assay Loading Reagent
    2.5
    Total volume
    5.0
    Final concentration (at 10x)
    Primers: 9 μM: Probe: 2 μM

    1. Seal the plate with the optical adhesive film.
      Important: For unused sample inlets, add 3.3 μl of sample mix and 2.7 μl of DNA-free water per inlet, for unused assay inlets, add 3.0 μl assay loading reagent and 3.0 μl of water.
  1. Preparing sample mixture
    Combine the components in the table below to make the Sample Mixture (scale up appropriately for multiple runs).
    Component
    Volume per Inlet (μl)
    TaqMan® Universal PCR Master Mix (2x)
    2.5
    20x GE sample loading reagent
    0.25
    cDNA (already diluted 1/5)
    2.25
    Total volume
    5.0

    1. Seal the plate with the optical adhesive film.
  1. Priming the 96.96 Dynamic ArrayTM in the Integrated Fluidic Circuit (IFC).
    1. Take a BioMark 96 x 96 Dynamic Array from its bag. Avoid touching the center of the chip.
      Control line fluid on the chip or in the inlets makes the chip unusable. This chip needs to be use within 24 h after opening the package.
    2. Take the 96.96 syringes with 150 μl of control line fluid and inject the 150 μl of control line fluid into each accumulator on the chip by introducing the syringe through the accumulators and pushing down the black O-ring. Use one syringe per accumulator. Avoid bending the pipette tip from the syringe.
    3. Place the chip into the Integrated Fluidic Circuit (IFC) controller HX, then select the Prime (136x) script program to prime the control line fluid into the chip. This program takes (~20 min).
  1. Chip Loading
    1. Load the chip within 60 minutes of priming to reduce the loss of pressure within the chip.
    2. Vortex thoroughly and at 300 x g, 4 °C for 5 min all assay and sample solutions from the 96 well-plates before pipetting into the chip inlets.
      Important: It is crucial to avoid forming any bubbles inside the chip inlets when loading the 10x assay mixes (prepared in step 5) on the chip inlets located on the left side and the 10x sample mixes (prepared on step 6) on the chip inlets located on the right side of the chip. To avoid bubbles, press the pipette only until the first stop. If bubbles appeared, pop them with a fresh sterile tip. Take care of cross-contamination or loss of reaction volume.
    3. Place the chip back into the IFC Controller HX, select the Load mix (163x) program to load the samples and assays into the chip. Selecting OK starts the loading. This program takes (~1 h and 30 min).
    4. While the Load Mix (136x) program is running select the “BioMark data collection Fluidigm” program from the computer.
    5. Select “double-click to warm up the lamp” from the program. The lamp will be ready in 20 min and the lamp indicator on the software should be in green.
    6. When the Load Mix (136x) program has finished, remove loaded chip from the IFC Controller HX.
  1. Using the data collection software.
    1. Double-click the data collection software icon on the desktop to launch the software.
    2. Click start a New Run to open the machine door.
    3. Check the status bar to verify that the camera temperature and the lamp are ready in green.
    4. Remove the blue plastic film just before placing the chip on the tray. Place the chip with the barcode facing the outside of the machine. Follow the “A” orientation.
    5. Click Load.
    6. Verify that the reader identifies properly the chip barcode and recognizes the chip type.
    7. Click Next.
    8. Click run file.
      1. Select new.
      2. Browse for a file location where you want your data to be stored.
      3.  Click Next.
    1. Amplification, Reference, Probes:
      1. Select application type: gene expression.
      2. Select passive reference: (ROX).
      3. Select assay: Single probe.
      4. Select probe types: FAM-MGB.
      5. Click Next.
    1. Click Browse to find thermal protocol file: GE 96 x 96 Standard v1.pcl
      PCR profile included:
      95 °C for 10 min
      40 cycles 95 °C for 15 sec and 60 °C for 60 sec
    2. Confirm auto expose is selected.
    3. Click Next.
    4. Verify the chip run information.
    5. Click Start Run. This program takes (~2 h).
  1. Result analysis Single-cell gene expression profiling represented as CT values.
    1. When the run has been completed, open the program “Fluidigm Real-time PCR analysis”.
    2. Open the chipRun file. bml to be analyzed.
    3. Select “Analysis view”.
    4. With the view of the results, set the following parameters for your analysis: the quality threshold, the baseline correction and CT threshold method.
    5. Click “Analyze”.
    6. The results are shown as CT values. The CT values can be seen in the software as a heat- map view, image view or as a table. Export all files with the “.cvs” extension.
    7. The data can be visualized and fully analyzed using Fluidigm real-time PCR analysis or any program for gene expression arrays.

Acknowledgments

We thank all the authors of this paper MacArthur et al. (2012) that participated in the single cell generation profiling as well as the data analysis. We gratefully acknowledge funding support by NIH (GM078465) and NYSTEM (C024410) to IRL. This work was also supported by an EPSRC Doctoral Training Centre grant (EP/G03690X/1) and an EPSRC 2011/12 Institutional Sponsorship Award (EP/J501530/1).

References

  1. MacArthur, B. D., Sevilla, A., Lenz, M., Muller, F. J., Schuldt, B. M., Schuppert, A. A., Ridden, S. J., Stumpf, P. S., Fidalgo, M., Ma'ayan, A., Wang, J. and Lemischka, I. R. (2012). Nanog-dependent feedback loops regulate murine embryonic stem cell heterogeneity. Nat Cell Biol 14(11): 1139-1147.

简介

该方案描述了如何使用Fluidigm Biomark?sup?9696动态阵列从单个小鼠干细胞进行高通量表达谱分析,使用多达96个定量PCR(qPCR)探针测定多达96个独立样品(相当于 9,216个反应)。 该动态阵列包含微流体通道,腔室和阀门的网络,其自动组装所有这些单独的PCR反应。 单细胞谱分析可以提供干细胞异质性的明确证据。 如果用户打算使用Biomark TM 48.48动态阵列或实验设计的引物与DNA结合染料如EvaGreen(Biotium 31000)结合,则很可能需要进行修饰。

图1.使用Fluidigm Biomark TM 动态的单细胞基因表达谱在小鼠干细胞分析中的工作流程。 此协议的主要基本步骤是:在96孔板上进行单细胞分选,特异性靶扩增,芯片加载,单细胞基因表达RT-PCR和数据收集。这种Fluidigm动态阵列集成流体电路芯片(IFC)包含微流控通道,腔室和阀门的网络,其自动组装单独的PCR反应。有关此特定芯片的更具体的信息,请访问 http://www.fluidigm。 com/single-cell-gene-expression.html。

材料和试剂

  1. 小鼠ES细胞(E14细胞系)(ATCC ,目录号:SCRC-1040 TM)
  2. 低EDTA(0.1mM EDTA)TE缓冲液(Teknova,目录号:T0227)
  3. 细胞一步法qRT-PCR试剂盒(Life Technologies,Invitrogen TM,目录号:11753-100)
  4. 用于研究的感兴趣的基因的Taqman基因表达测定(Applied Biosystems)
    注意: 测定是使用TaqMan MGB(小沟结合剂)探针的基于5'核酸酶化学的探针和引物组。在此处搜索您的Taqman基因表达测定: https://bioinfo.appliedbiosystems.com/genome-database/gene-expression.html
    1. 选择您正在进行的实验类型。单击基因表达
    2. 选择所需的测定类型。点击所有基因表达测定
    3. 选择您要定位的物件。点击鼠标,因为我们的样品是Mouse ES细胞。
    4. 输入目标信息。添加基因符号或entrez基因符号或entrez基因要分析其表达的基因的ID。
  5. 96.96用于基因表达的动态阵列芯片(Fluidigm,目录号:BMK-M-96.96)
  6. 基因表达96.96动态阵列样品& 装载试剂盒(Fluidigm,目录号:85000802),其包含:
    1. 两管的2x测定加载试剂(Fluidigm,目录号:85000736)
    2. 两管20x GE样品加载试剂(Fluidigm,目录号:85000746)
    3. 20个对照线流体注射器(150μl)
  1. 通用PCR主混合物(Applied Biosystems,目录号:4304437)
  2. Dulbecco's磷酸盐缓冲盐水(DPBS)(Life Technologies,目录号:14190-136)
  3. TryPLE快速溶液(Life Technologies,目录号:12605-010)
  4. 4',6-二脒基-2-苯基吲哚(DAPI)(Life Technologies,目录号:D1306)
  5. 碘化丙啶(PI)(Life Technologies,目录号:P-3566)
  6. 2x Assay Loading Reagent(Fluidigm,目录号:85000736)
  7. 2x TaqMan?Universal PCR Master Mix(Applied Biosystems,目录号:4304437)
  8. 20x GE样品加载试剂(Fluidigm,目录号:85000746)

设备

  1. 96孔半裙板,0.2ml孔,直立的侧面(美国科学,目录号:1402-9200)
  2. 光学粘合剂膜(Applied Biosystems,目录号:4311971)
  3. PCR-8管条& Strip Camps,natural(USA Scientific,目录号:1402-2500)
  4. TipOne0.1-10μl延伸长度过滤嘴(美国科学,目录号:1120-3810)
  5. TipOne 101-1,000μl长度延长的过滤嘴(美国科学,目录号:1122-1830)
  6. TipOne1-200μl分级过滤嘴(美国科学,目录号:1120-8810)
  7. 荧光激活细胞分选仪(FACS Aria II; BD Biosciences,目录号:643756)
  8. 多通道移液器(10-100μlEppendorf Research ,目录号:38-3122000043;0.5-10μl目录号:38-3122000019)
  9. 热循环仪(Veriti?sup?96-well Applied Biosystems,目录号:PN 4375786)
  10. 集成流体电路IFC控制器
  11. MX模型填充并加载48.48个芯片
  12. HX模型填充和加载96.96芯片。
  13. 离心机(Eppendorf,目录号:5810)

程序

  1. 逆转录特异性靶扩增(RT-STA)主混合物制备。
    1. 解冻CellsDirect TM 一步法qRT-PCR试剂盒的CellsDirect 2x反应混合物。所有步骤应在冰上进行,试剂应在使用前立即冷冻和/或解冻。
    2. 将65μl的2x Reaction Mix转移到PCR-8-Tube Strip的每个管中。
    3. 使用多通道移液器从PCR-8管柱中加入5μl2x反应混合物到96孔板的每个孔中。
      由于来自单个细胞的RNA的量是很少的象形图,我们不执行传统的RNA分离方案。相反,将分选的细胞直接引入2x反应混合物中。为了确保最佳的样品保存,此主混合物包含CellDirect 2x反应混合物(CellsDirect一步qRT-PCR试剂盒)和SUPERase-In。该酶保护从裂解细胞提取的RNA免受可能存在于RT-STA主混合物中的任何RNA酶的影响。

  2. 细胞分选
    1. 用TryPLE Express解决方案分离小鼠ES细胞。 轻轻吸出细胞培养基,用5-6ml新鲜的1×DPBS洗涤细胞,以去除可抑制TryPLE Express溶液活性的血清。
    2. 吸出1×DPBS。
    3. 将1ml预热的TryPLE Express溶液直接加入到细胞培养板中,并将板在37℃,5%CO 2下孵育3-5分钟。
    4. 取出细胞培养板,轻轻拍打或摇动,然后加入10毫升新鲜细胞培养基与10%(v/v)FBS停止胰蛋白酶活性。 用移液管混合细胞,并将1×10 6个细胞转移到15ml锥形管中。 以300×g离心,4℃5分钟。
    5. 吸出细胞上清液,并在补充有5%(v/v)FBS的1ml 1x DPBS中重悬1×10 6个细胞。
    6. 保持1×10 <6> 细胞在冰上,然后根据您有兴趣研究的细胞群体,用合适的表面标记标记细胞。
    7. 每1×10 6个细胞加入10微升的DAPI(> 200纳克/毫升)或10微升的PI(5微克/毫升)到单细胞悬浮液含有1×10
    8. 在FACS Aria II中进行细胞分选和用FACS Diva软件进行数据分析。
    9. 当在排序参数中对单元格排序单元格时。
    10. 将单个细胞直接分入含有5μlCellsDirect 2x反应混合物(组分细胞直接一步法qRT PCR试剂盒,Invitrogen)的96孔板的孔中。我们估计细胞分选到孔中后的反应终体积为6μl 注意:将细胞分类到用于RT-STA PCR的相同平板中。始终将井板保持在冰上。
    11. 在300×g离心,4℃5分钟96孔板,让细胞进入CellsDirect 2x反应混合物。
    12. 用光学粘合膜密封板。
    13. 将板存储在-80°C。 这个过夜冷冻步骤允许更好的溶解效率
  3. 制备特异性靶标扩增(RT-STA)主混合物。
    1. 为96孔板制作excel文件模板,并使用特定的20x Taqman测定探针标记每个细胞。
    2. 解冻20x Taqman测定,并根据每个20x Taqman测定探针的excel文件模板中的位置将其存放在96孔板中。
    3. 在冰上用CellsDirect 2x反应混合物中分选的细胞解冻板。
    4. 准备测定混合在1.5毫升eppendorf管中。 池所有Taqman实时测定,将在单细胞实时PCR分析,您以前在步骤b中沉积在96孔板上。 在混合物中使用的Taqman测定的最终浓度将为0.2倍。 使用TE缓冲液稀释Taqman测定。 这是0.2x测定混合物。
      组件
      每反应加入的体积(μl)
      最终浓度
      20x Taqman基因表达测定1
      1.5
      0.2x
      20x Taqman基因表达测定2
      1.5
      0.2x
      ...
      ...
      ...
      TE缓冲区
      高达150μl

      TOTAL
      150μl

    5. 通过组合以下试剂制备样品RT-STA Master Mix:
      组件
      体积(μl)
      0.2x测定混合物
      2.5
      SuperScript TM RT/Platinum ® Taq Mix
      0.2
      TE缓冲区
      1.3
      TOTAL
      4
      注意:为了克服移液限制,对所有正在测试的样品创建批量解决方案非常重要。
    6. 将4μlRT-STA主混合物加入到每个孔中,其中已经包含浸入到CellsDirect 2x反应混合物中的分选细胞。
    7. 用光学粘合剂膜密封板。 让板在冰上5分钟,同时你编程热循环仪 在运行逆转录和特异性靶扩增之前,验证来自96孔板的每个孔具有根据下表的组分和体积。 您的最终体积必须为9μl。
      零件 体积(μl)
      CellsDirect 2x反应混合物(此试剂已在分选的细胞的平板中)
      5.0
      0.2x测定混合物
      2.5
      SuperScript TM RT/Platinum ® Taq Mix
      0.2
      TE缓冲区
      1.3
      TOTAL
      9

  1. 逆转录和特异性靶扩增。
    1. 将96孔板与RT-STA主混合物放在热循环仪中。
      50℃15分钟(将RNA逆转录成cDNA的步骤)
      95℃2分钟(使RT酶失活并激活Taq的步骤)
      22个循环的95℃15秒和60℃4分钟(STA特异性靶扩增)
      保持在4°C
    2. 根据这些参数运行样品PCR:
      注意:我们筛选了不同数目的循环,我们发现for 单细胞分析22个循环在芯片中给我们更好的结果 分辨率。
    3. 用TE缓冲液稀释所得的cDNA产物1:5 可以在1:2和1:5之间稀释。
  1. 准备10x测定
    在无DNA罩中,使用下表中的体积(对于多次运行适当放大),制备具有5μl等分试样的10×测定的96孔板。 将20x TaqMan测定,每孔一个,加入到96孔板中,在与excel文件模板相同的位置。
    组件
    每次进样量(μl)
    20x Taqman 基因表达测定(Applied Biosystems) 2.5
    2x分析加样试剂
    2.5
    总量
    5.0
    最终浓度(10x)
    引物:9μM:探针:2μM

    1. 用光学粘合膜密封板。
      重要:对于未使用的样品入口,对于未使用的测定入口,加入3.3μl样品混合物和2.7μl无DNA水,加入3.0μl测定上样剂和3.0μl水。
  1. 准备样品混合物
    合并下表中的组分以制备样品混合物(适用于多次运行)。
    组件
    每次进样量(μl)
    TaqMan Universal PCR Master Mix(2x)
    2.5
    20x GE样品加载试剂
    0.25
    cDNA(已稀释1/5)
    2.25
    总量
    5.0

    1. 用光学粘合剂膜密封板。
  1. 在集成流体电路(IFC)中启动96.96动态阵列 TM
    1. 从BioMark 96 x 96动态阵列袋中取出。 避免接触芯片的中心。
      在芯片上或在入口中的控制线流体使得芯片不可用。 该芯片需要在打开包装后24小时内使用。
    2. 取带有150μl对照线流体的96.96注射器,并通过将注射器引导通过蓄能器并向下推动黑色O形环,将150μl控制线流体注入芯片上的每个蓄能器。 每个蓄电池使用一个注射器。 避免将移液器尖端从注射器弯曲。
    3. 将芯片放入集成流体电路(IFC)控制器HX,然后选择Prime(136x)脚本程序,将控制线流体注入芯片。此程序需要(〜20分钟)。
  1. 芯片加载
    1. 在启动后60分钟内装入芯片,以减少芯片内的压力损失
    2. 在移液进入芯片入口之前,在4℃下彻底涡旋5分钟,来自96孔板的所有测定和样品溶液,并在300℃下 /> 重要的是,当将位于左侧的芯片入口上的10x测定混合物(在步骤5中制备)和10x样品混合物(在步骤6上制备的)装载在芯片入口上时,避免在芯片入口内形成任何气泡是至关重要的芯片入口位于芯片的右侧。为了避免气泡,请按移液器直到第一次停止。如果出现气泡,用新鲜的无菌尖端弹出。注意交叉污染或反应体积损失。
    3. 将芯片放回IFC控制器HX,选择加载混合(163x)程序将样品和分析装载到芯片中。 选择OK开始加载。 此程序需要(〜1小时30分钟)。
    4. 当载入混合(136x)程序运行时,从计算机选择"BioMark数据收集Fluidigm"程序。
    5. 从程序中选择"双击以预热灯"。 灯泡将在20分钟后准备就绪,软件上的灯泡指示灯应为绿色
    6. 当加载混合(136x)程序完成后,从IFC控制器HX中卸载加载的芯片。
  1. 使用数据收集软件。
    1. 双击桌面上的数据收集软件图标以启动软件。
    2. 单击开始新运行以打开机器门。
    3. 检查状态栏以验证相机温度和指示灯是否已准备好为绿色。
    4. 在将芯片放在托盘上之前,取下蓝色塑料膜。 将条码朝向机器外部的芯片放置。 按照"A"方向。
    5. 单击加载。
    6. 验证读取器正确识别芯片条形码并识别芯片类型。
    7. 单击下一步。
    8. 单击运行文件。
      1. 选择新。
      2. 浏览要存储数据的文件位置。
      3.  点击下一步。
    1. 扩增,参考,探针:
      1. 选择应用程序类型:基因表达。
      2. 选择被动参考:(ROX)。
      3. 选择测定:单探针。
      4. 选择探头类型:FAM-MGB。
      5. 单击下一步。
    1. 单击浏览查找热协议文件:GE 96 x 96标准v1.pcl
      PCR配置文件包括:
      95℃10分钟
      40个循环95℃15秒和60℃60秒
    2. 确认已选择自动曝光。
    3. 单击下一步。
    4. 验证芯片运行信息。
    5. 单击开始运行。 此程序需要(〜2小时)。
  1. 结果分析单细胞基因表达谱表示为CT值。
    1. 当运行完成后,打开程序"Fluidigm实时PCR分析"。
    2. 打开chipRun文件。 bml进行分析。
    3. 选择"分析视图"。
    4. 根据结果,设置以下参数用于分析:质量阈值,基线校正和CT阈值方法。
    5. 点击"分析"。
    6. 结果显示为CT值。 在软件中可以看到CT值作为热图视图,图像视图或表格。 导出扩展名为".cvs"的所有文件。
    7. 可以使用Fluidigm实时PCR分析或基因表达阵列的任何程序使数据可视化和完全分析。

致谢

我们感谢参与单细胞生成分析以及数据分析的本文的所有作者MacArthur等人(2012)。 我们衷心感谢NIH(GM078465)和NYSTEM(C024410)对IRL的资助。 这项工作还得到了EPSRC博士培训中心资助(EP/G03690X/1)和EPSRC 2011/12机构赞助奖(EP/J501530/1)的支持。

参考文献

  1. MacArthur,BD,Sevilla,A.,Lenz,M.,Muller,FJ,Schuldt,BM,Schuppert,AA,Ridden,SJ,Stumpf,PS,Fidalgo,M.,Ma'ayan, 和Lemischka,IR(2012)。 Nanog依赖性反馈环调节小鼠胚胎干细胞异质性。 Nat细胞生物学 14 11):1139-1147
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sevilla, A. (2013). Single-cell Gene Expression Profiling of Mouse Stem Cells With Fluidigm BiomarkTM Dynamic Array. Bio-protocol 3(9): e692. DOI: 10.21769/BioProtoc.692.
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Mikael Kubista Mikael
TATAA Biocenter
Guys, you may be interested in the single cell analysis workflow:

Anders St?hlberg, Vendula Rusnakova, Amin Forootan, Miroslava Anderova, Mikael Kubista
RT-qPCR work-flow for single-cell data analysis
Methods 59, 80-88 (2013)

(available on www.tataa.com if you don't have access).
12/27/2013 2:22:17 PM Reply
Qingqing Wei
China Agricultural university
Thank you for sharing this protocal.
I have read the user manual of The CellsDirectTM One-Step qRT-PCR kit and have a question to consult.
The kit contains Lysis Solution (Resuspension buffer and Lysis Enhancer), but The 2x Reaction Mix provided in this kit only consists of a proprietary buffer system, MgSO4, dNTPs, and stablizers. According to this protocol, if the single cell does not need to lyze?
12/6/2013 4:36:38 AM Reply
Ana Sevilla
Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, USA

Hi Quingqing
For the lysis, Fluidigm technical support suggested to use only the 2X Reaction buffer from the CellsDirectTM One-Step qRT-PCR kit and I never got issues related with incomplete lysis.

12/17/2013 5:34:46 AM


Maria Solano
IDIBELL
Thank you for posting this protocol. This is very nice for us out there trying to set the assay up.

How many technical repeats did you do per Taqman Assay primer pair?

How many technical repeats did you do per Biological replicate (ie cell)?



My understanding is that both Taqman Assay primer pair and the Superscript are can be used 4x more for the Fluidigm assay. How much volume of each reagent did you purchase for your experiment?

9/11/2013 12:42:11 PM Reply
Ana Sevilla
Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, USA

Hi Maria.
In the 96 Fluidigm Chip you have the option to interrogate 96 Taqman Assay Primers.
For single cell assays I would recommend you to do more biological replicates rather than technical replicates. I have run twice the same cDNA in two different Fluidigm Chips and the results were the same. The technique is pretty robust.
In our paper we interrogated 96 cells (biological replicates) per condition. For single cell analysis the more biological replicates you can afford the more robust statistic results you will get.
The size of the Taqman Assay Primers you will have to purchase will depend on the number of Chips you would like to run.

9/15/2013 4:04:24 PM