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Sep 2019

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Single-cell qPCR Assay with Massively Parallel Microfluidic System
大规模平行微流控系统用于单细胞qPCR检测   

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Abstract

The single-cell transcriptome is the set of messenger RNA molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Due to sensitivity shortages, most of genetic studies use bulk samples, providing only the average gene expression. Single-cell technologies have provided a powerful approach to a more detailed understanding of the heterogenic populations and minority cells. However, since it is still a quite novel technique, standardized protocol has to be established. Single-cell qPCR, although partly limited by the number of genes, is relatively simple to analyze. Therefore, its use is accessible without the necessity to recourse to complex bioinformatics analyses. The main steps for single-cell qPCR, as illustrated in this protocol, are composed by single-cell isolation, cell lysate, cDNA reverse-transcription synthesis, amplification for cDNA library generation, and finally, quantitative polymerase chain reaction.

Keywords: Single-cell (单细胞), Transcriptomics (转录组学), Single-cell qPCR (单细胞qPCR), mRNA (mRNA), Microfluidic system (微流控系统), Cell heterogeneity (细胞异质性)

Background

The single-cell transcriptome is the complete set of messenger RNA (mRNA) molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Hence, it is the source of the biological heterogeneity, in which the cells from the same environment are similar -but not identical- to the other ones.

Due to sensitivity shortages, most of genetic studies use bulk samples, with hundreds to thousands of cells, providing only the average gene expression. This vastly limits the study of minor populations of cells, which may poses specific properties, such as drug resistance or metastatic ability in the case of cancer. Single-cell technologies enable the analysis of the transcriptome at single cell level, unraveling the complexity of heterogenic populations. These technologies have been applied not only in cancer but in many cell biology studies, including adult tissues, stem cells, immune cells; as well as other fields such as microbiology and virology (Wen and Tang, 2016; Karaiskos et al., 2017; Rato et al., 2017; Woyke et al., 2017; Zheng et al., 2017).

There are two main single-cell transcriptome approaches: mRNA sequencing and quantitative polymerase chain reaction. Although mRNA sequencing enables the study of the whole transcriptome without prior knowledge, manipulating an extremely large amount of genes can be overwhelming for novel researchers in the field; moreover high levels of bioinformatics are required (Prieto-vila et al., 2018). To counterbalance, single-cell qPCR analysis is limited to a certain number of genes, but can be analyzed as normal qPCR, being more approachable.

In our previous research, we aimed to study a drug resistant subpopulation of cells by using single-cell qPCR in breast cancer cell lines. With that purpose, we used massively parallel single-cell amplification, that allowed us the study of 96 genes at single cell level to 96 cells per run (Prieto-Vila et al., 2019). This is one of the most commonly used techniques for single-cell qPCR in which reactions are carried in specific 96-well plates, where individual samples are reverse transcribed at the same time. The C1 nanofluidics along with the Fluidigm systems, are the most commonly used systems for sc-qPCR. C1 machine is a hydrodynamic cell trap chip, formed by a net of channels where the cells are eluted and single cells are fiscally separated their size. Thanks to this system, rarely doublets are found. The 96 single cells are then eluded into 96 individual wells. On these wells lysis, reverse transcription, and pre-amplification for qPCR are carried in parallel using a very small reaction volume (Ziegenhain et al., 2017). Following, massive parallel qPCR is done by analyzing the expression of 96 genes in the 96 individual cells in a matrix-manner chip by the Fluidigm system.

In the present protocol, we provide a single-cell transcriptome analysis protocol, including experimental details to perform single-cell qPCR with the Fluidigm systems.

Materials and Reagents

  1. Pipette tips 1,000 μl/200 μl/20 μl (Sorenson Bioscience, catalog numbers: 34000 / 14220 / 15020 )
  2. Low binding centrifuge tubes 1.5 ml (Watson BioLab, catalog number: pk-15 c-500 )
  3. Rainin LTS tips 10 μl (Rainin, catalog number: 30389228 )
  4. qPCR 96-well plates (Applied Biosystems, catalog number: 4346907 )
  5. Film for qPCR plates (Applied Biosystems, catalog number: 4311971 )
  6. Nuclease-Free water (Ambion, catalog number: AM9932 )

For cell culture
  1. Cell culture 100 mm dishes (Thermo Fisher Scientific, catalog number: 172931 )
  2. 15 ml conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 339650 )
  3. Desired cell line, in this case we used the breast cancer cell lines MDA-MB-231 and MCF7
  4. RPMI 1640x basic medium (Thermo Fisher Scientific, catalog number: C11875500BT )
  5. Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106 )
    Note: FBS should be heat inactivated at 56 °C for 30 min before use.
  6. Antibiotic/antimitotic solution (Thermo Fisher Scientific, catalog number: 15240062 )
  7. Phosphate buffer Saline (PBS) (DS Pharma Biomedical, catalog number: DSBN200 )
  8. TrypLE Express (Thermo Fisher Scientific, catalog number: 12604013 )
  9. Trypan Blue Solution 0.4% (Gibco, catalog number: 15250-061 )
  10. Neubauer Improved C-Chip (NanoEnTek, catalog number: DHC-N01 )
  11. 0.5 mol/L EDTA (Nacalai Tesque, catalog number: 06894-14 )
  12. Initial cell suspension buffer (see Recipes)

For single cell isolation and pre-amplification
  1. C1 Single-Cell Auto Prep IFC for Preamp (10-17 μm) (Fluidigm, catalog number: 100-5749 )
    Note: There are several channel size plates to adjust to the cell size. See notes for more information.
  2. 20x gene specific Assays (Taqman)
  3. C1 Single-Cell Auto Prep Reagent Kit (Fluidigm, catalog number: 100-5319 )
  4. Ambion single cell-to-Ct qRT-PCR Kit (Thermo Fisher Scientific, catalog number: 4458237 )
  5. Live/Dead Kit for mammalian cells (Life Technologies, catalog number: L-3224 ) (see Recipes)
  6. Lysis mix (see Recipes)
  7. Reverse transcription final mix (see Recipes)
  8. 0.2x Pooled Taqman primers (see Recipes)
  9. Pre-amplification mix (see Recipes)

For single cell qPCR
  1. Fluidigm 96.96 quantitative PCR Dynamic Array microfluidic chips (Fluidigm, catalog number: BMK-M-96.96GT )
  2. Taqman Universal PCR Master Mix 2x (Applied Biosystems, catalog number: PN 4304437
  3. 20x GE Sample Loading Reagent (Fluidigm, catalog number: PN 100-7610 )
  4. 2x Assay Loading Reagent (Fluidigm, catalog number: PN 100-7611 )
  5. 10x Assays for single-cell qPCR (see Recipes)
  6. Sample pre-mix (see Recipes)

Equipment

  1. Neubauer chamber
  2. Micropipettes (P2, P10, P20, P200, P1000)
  3. Multichannel pipette P10 with high precision (Rainin, catalog number: 17013802 )
  4. Cell culture incubator: 37 °C and 5% CO2 (Panasonic Healthcare, catalog number: MCO-170AICUVH-PJ )
  5. Clean bench (Panasonic, catalog number: MCV-131BNF-PJ )
  6. Centrifuge for 1.5 ml tubes (Eppendorf, model: 5418R )
  7. Centrifuge for 15 ml tubes (Tomy, model: Ax-511 )
  8. Centrifuge for 96-well plate (Sigma, model: 4-16KS )
  9. Vortex (Scientific Industries, model: Vortex-Genie 2 )
  10. Fluorescent confocal microscope (Keyence, model: BZ-X700 )
  11. Refrigerator and Freezer (any company’s product should be fine)
  12. C1 Auto Prep System (Fluidigm)
  13. IFC Controler HX (Fluidigm)
  14. BioMarkHD system for real-time qPCR (Fluidigm)

Software

  1. Fluidigm Real-Time PCR Analysis Software v.2.1.3
  2. Fluidigm Data Collection Software v.2.1.3
  3. Excel
  4. R (version 3.3.2)

Procedure

Single-cell qPCR protocol can be mainly divided into 4 fractions: The first one comprises cell culture and cell suspension preparation. The second and third are mainly done by the C1 integrated fluidic circuits, which allows single cell separation, cell lysis and pre-amplification. The final fraction is the single real time qPCR, which is done by the BioMarkHD system. Those steps are represented in Figure 1.


Figure 1. Scheme of single-cell qPCR protocol

  1. Sample preparation
    This protocol commences with cell culture already ongoing.
    1. When the cells reach 70% of confluence are ready to collect.
    2. Remove the medium and wash the dish with PBS once.
    3. Add 1 ml of TrypLE Express.
    4. Place the dishes in the 37 °C incubator, and incubate for 3-5 min until the cells detach.
    5. Add 4 ml of pre-warmed complete medium at 37 °C to stop the reaction.
    6. Pipette delicately to ensure the complete detach of all the cells but without damaging the cells.
    7. Transfer the cells into 15 ml tube.
    8. Centrifuge at 300 x g 4 °C for 5 min.
    9. Aspirate the medium and add 5 ml of PBS.
    10. Take apart 100 μl to count the cells.
    11. Centrifuge the 15 ml tube at 300 x g 4 °C for 5 min.
    12. While centrifuging, mix the separated cells at a proportion of 1:1 with Trypan blue solution.
    13. Insert 10 μl of the mixt into Neubauer chamber.
    14. Count the total cell number with the microscope (including dead cells).
    15. Remove PBS.
    16. Re-suspend the cells at 300 cells/μl (3 x 105 cells/ml) in Initial cell suspension buffer (Recipe 1) and keep on ice.
      Note: An amount of at least 10,000 cells are recommended to obtain a complete capture, we observed that less than that amount leaves multiple channels with cells unattached in the IFC plate.

  2. Single-cell separation
    1. Take all the necessary reagents from -20 °C, where are stocked, and let them thaw on ice for 30 min before starting the experiment. Just before starting, take the reagents kept at 4 °C and put them on ice during its use.
      Caution: The plate must not be placed on ice.
    2. Remove the black plastic situated at the button of the integrated fluidic circuits (IFC) plate.
    3. Before starting, turn on the C1 machine since it takes some time to start up (about 3 min).
    4. Pipette several reagents following this scheme:


      Figure 2. Representation of IFC plate indicating the wells to add the different reagents and solutions. Same number indicates same solution and volume. The wells covered in grey, represent the section of the chip that is covered by a film of paper, which at the end of the experiment will contain, each single-cell sample pre-amplified ready to use in sc-qPCR.
      ①. Add 200 μl of Harvest reagent. This well is like a button with a plastic lid on an iron spindle. To apply the reagent, press slightly with the tip, and once slips down, put the tip on the space. Then add slowly the reagent. As can be seen in Figure 3.
      ②. Add 20 μl of Harvest reagent.
      ③. Pipette 15 μl of Blocking reagent.
      ④. Add 20 μl of Preloading reagent.
      ⑤. Add 20 μl of Wash Buffer.


      Figure 3. Scheme of Harvest reagent (200 μl) application in the IFC plate

    5. Insert the plate in the IFC machine with the barcode facing outside.
      Note: If the bottom of the plate is dirty, the machine will not detect it and will give an error. If so, wipe the bottom with ethanol 70% and place it again.
    6. Run the mode STA:Prime (1782x/1783x/1784x) – according to the selected size of channels of the chip. This procedure takes 10-12 min.
      While priming, prepare the cell mixture.
    7. Prepare the cell mixture: 30 μl of cell suspension and 20 μl of suspension reagent.
      Note: Less amount of cell mix can be prepared as long as the ratio is maintained. For instance, 6 μl and 4 μl.
    8. After priming is finished, click eject and take out the plate.
    9. Remove the leftovers of C1 blocking reagent with a P200 pipette (wells are indicated in Figure 2-③).
    10. Insert the cells and Live/Dead staining solutions, as follows:


      Figure 4. Representation of IFC plate indicating the wells to add the different reagents and solutions
      ①. Add 6 μl of cell mixture.
      Note: Do not vortex the cells. Pipette them before the addition.
      ②. Add 20 μl of Live/Dead staining solution (Recipe 2).

    11. Place the IFC plate into the C1 system.
    12. Select STA: Cell Load&Stain (1782x/1783x/1784x)–according to the selected size of channels of the chip. It takes 30-65 min, depending on the cell size of the plate.
    13. Meanwhile, prepare the Lysis Mix, RT Mix and PreAmp Mix (Recipes 3, 4 and 5).
    14. When Loading is complete, withdraw the plate from the C1 and take pictures of the cells with Keyence microscope. Using the Phase contrast at 10x, channels and fluorescence-stained cells can be easily observed on the central part of the plate, colored in grey color in Figure 4. Also each well number can also be observed.
    15. Write down the channels with dead cells, empty channels and channels with more than one cell to be discarded a posteriori (Figure 5).


      Figure 5. Example of Live/Dead staining. Green cells are living cells while read are dead, or highly damaged cells. Moreover, right chamber shows a doublet, and left one shows a clear single cell.

  3. Cell lysis, reverse transcription and pre-amplification.
    1. Add the reagents following the scheme in Figure 6:


      Figure 6. Representation of IFC plate indicating the wells to add the different reagents and solutions
      ①. Add 180 μl of Harvest reagent. Carefully move the plate from side-to-side to ensure that the reagent covers all the surface.
      ②. Add 7 μl of Lysis Final Fix.
      ③. Add 7 μl of RT Final mix.
      ④. Add 24 μl of Pre-amplification Mix (Recipe 6).

    2. Place the plate into the C1 system.
    3. Select STA: PreAmp (1782x/1783x/1784x)–according to the selected size of channels of the chip- and choose the finish hour. We usually leave it overnight and collect it next morning to proceed with the experiments. The process itself takes 5-6 h approximately.
    4. When STA: Preamp process has finished, select EJECT and remove the IFC plate.
    5. Using a new PCR 96-well plate, add 25 μl of C1 dilution reagent in each well.
    6. Remove the papers than protect the output wells on IFC plate (those covers are represented in grey color in Figures 2, 4 and 6).
    7. Using an eight-channel micropipette (Rainin), transfer the samples onto the 96-well plate.
      Note: The amount of sample should be close to 3 μl, but to ensure, please set the micropipette at 4 μl.
    8. Transfer the samples using this specific order, described in Figure 7.
      Note: Using 8-channel micropipette, and taking in consideration that IFC plate has 16 well per lane, it has the specific size to pic up every-other well.


      Figure 7. Scheme to illustrate the order of well transfer from IFC plate to PCR 96-well plate

    9. Vortex the 96-well plate and spin down. At this point, the samples can be frozen at -20 °C and be kept for few weeks before use, in that case, seal the plate with a PCR-plate film.

  4. Single-cell qPCR
    1. Take all the necessary reagents from -20 °C, where are stocked, and let them thaw on ice for 30 min before starting the experiment. Just before starting, take the reagents kept at 4 °C and put them on ice during its use.
      Caution: The plate must not be placed on ice.
    2. Turn on the Standard 96-well Thermal Cycler, because it takes some time to start up.
    3. Remove the blue protective film from the bottom of the 96.96 chip.
    4. Using the syringe from the kit, inject the control line fluid in the two big central wells, pressing between the cross (The cross can be seen, marked with an arrow in Figure 8).
      Note: The oil leaks easily, so be careful when managing the syringe.


      Figure 8. Schematic representation of the 96.96 quantitative PCR Dynamic Array microfluidic chip. The arrow shows the space in where the line fluid should be applied.

    5. Insert the plate (with the barcode facing the outside).
    6. Select the option Prime(136x). This process takes approximately 20 min.
    7. Meanwhile, prepare the Pre-mix sample (Recipe 8).
    8. In a new PCR 96-well plate, add 3.3 μl of sample pre-mix in each well.
    9. Add 2.7 μl of cDNA sample with a multichannel micropipette (Sample obtained at Step C9) making a total volume of 6 μl in each inlet.
    10. Seal with PCR-plate film.
    11. Prepare the Taqman assays Mix (Recipe 7) and seal the plate with PCR-plate film.
    12. Vortex both plates for 10 sec and spin down.
    13. When priming is complete, collect the chip.
    14. Transfer the samples and the Taqman assays. In the following order (Figures 9 and 10):


      Figure 9. Scheme of 96.96 chip showing in yellow color the inlets to insert Taqman assays and, in green, the inlets to insert sample premix
      ①. Add 5 μl of Taqman assays in the yellow inlets, using the following order (Figure 10A) to transfer from qPCR 96-well plate to IFC chip.
      ②. Add 5 μl of Sample Premix, using the same order (Figure 10A) in the green inlets.
      Notes:
      1. The inlet wall is not horizontal, but has a diagonal angle. To ensure better application; the best option is insert the tip inclined, as is illustrated in Figure 10B.
      2. Avoid bubbles when pipetting. However, if there are bubbles but they move, means that they are on the surface, and then they will not interfere in the analysis. If bubbles do not move, with a micropipette tip remove the bubble.


      Figure 10. Scheme of order and methodology to transfer samples into 96.96 chip. A. Scheme to illustrate the order of well transfer from PCR plate to 96.96 chip. B. Scheme of sample application in 96.96 inlets.

    15. Insert the chip into the IFC Controller HX and run the script Load Mix (136x).
    16. While running the Load script, turn on the computer connected to the BioMarkHD system for real-time qPCR.
    17. Run the software Biomark data collection.
      Double click to turn on the option “lamp” to start up. This process takes approximately 20-30 min.
    18. When the Loading finish, bring the chip to BioMarkHD system and follow these steps:
      1. Click “Start a new run”.
      2. Insert the chip with the bar code facing outside.
        Select: Load
             →Next
      3. Select the settings of the 96.96 chip.
        1. Choose the name and the folder where you want to save the document.
          Next
        2. Application type: Single probe.
        3. Probes: FAM_MGB
          Next
        4. Protocol: GE 96x96 Standard v1.
          Next
      4. Start the run.
    19. Once it finishes remove the plate, close the software and shut down the computer.

Data analysis

The data analysis procedure refers to our previous report (Prieto-Vila et al., 2019).

  1. Remove dead/damaged cells
    1. After running the qPCR, collect the raw data to be analyzed with the software Biomark.
    2. Use the settings Linear (derivative) and User (detectors) settings to generate Ct values for each gene.
    3. Remove all values with confidence lower than 0.2.
    4. Export the data to excel file.
    5. Using the data obtained in Step B14 from the protocol (pictures of Live/Dead staining), remove all the cells that were stained red (dead), chambers with more than one cell, or none.
    6. Remove cells whose housekeeping genes have Ct values lower than 16. In our case, we used both BACTIN and GAPDH.

  2. Data normalization
    Normalization can be conducted by two systems:
    1. With the objective to remove the variation between assays, normalization can be done using a housekeeping gene; in our case, we utilized GAPDH. The normalization was calculated by the formula: 2-ΔΔCT.
    2. Another broadly used methodology in single-cell analysis is the normalization by log2Ex, which expresses the transcript levels above the background in log base 2 (Livak et al., 2013). In our case, we set the LOD (limit of detection) to 26, as it was the average of the lowest detectable Ct value for each gene. This can calculated by the formula: Log2Ex = LOD (Limit Of Detection) Cq – Cq [Gene].
    Notes:
    1. Both normalizations can be done with Excel.
    2. For a more detailed protocol containing examples, please refer to this manual: “Singular: Analysis Toolset” (P/N 100-5066) https://www.fluidigm.com/binaries/content/assets/fluidigm/singular-analysis-toolset-3.5/singular-analysis-toolset-3.5-userguide.pdf.

  3. Several statistical analysis can be done for single-cell qPCR
    Probably the most common statistical analysis for single-cell are t-SNE Plot, Heatmap and ViolinPlots.
    For Heatmap we used the software Partek Genomics Suite Software; while both t-SNE and Violin Plot were done by the free software R using the package “ggplot2”.

Notes

  1. For single-cell analyses, it is considered that rather than do replicates, which is also physically impossible, is more important to increase the sample number. Thus, we ran each sample twice. Analyzing a total of 192 individual cells per sample.
  2. There are three sizes of C1 IFC plates to adjust the channel to the different sizes of cells. Prior to choose the size of the plate, we calculated the cell size by taking pictures of the cells after being detached, and measuring the size by Keyence software. The available sizes are: S (5-10 μm), M (10-17 μm), L (17-25 μm). As a reference, we used M size for MDA-MM-231 and MCF7 cells; and we have run hepatocytes in L size plate (Katsuda et al., 2019).
  3. One of the most important steps is single-cell isolation. During this process, cells are isolated from their local environment, and thus, keeping those cells alive and in good condition for proper further analyses is indispensable (Van Den Brink et al., 2017). Please manage them carefully.
  4. This protocol has been done following the Fluidigm system manufacture’s protocols (PN-100-6177 J1 and PN 68000130 E1), which can be found at: https://www.fluidigm.com/binaries/content/documents/fluidigm/resources/c1-taqman-pr-100%E2%80%906117/c1-taqman-pr-100%E2%80%906117/fluidigm%3Afile and https://www.fluidigm.com/binaries/content/documents/fluidigm/resources/96.96-ge-taqman%E2%80%90std-qr-68000130/96.96-ge-taqman%E2%80%90std-qr-68000130/fluidigm%3Afile respectively.

Recipes

Note: All the recipes, unless otherwise stated, are the reagent necessary for one run.

  1. Initial cell suspension buffer
    Components Volume
    Components Volume
    EDTA (5M)
    0.1 ml
    FBS
    50 μl
    PBS
    10 ml
    *This solution can be used multiple times and should be stored at 4 °C.
  2. LIVE/DEAD Cell Staining solution (Total 1253.13 μl)
    Components
    Volume
    Cell Wash Buffer (C1 Single-Cell Auto Prep Reagent Kit)
    1,250 μl
    Ethidium homodimer-1 (Live/Dead kit)
    2.5 μl
    Calcein AM (Live/Dead kit)
    0.63 μl
  3. Lysis Mix (Total 18.1 μl)
    Components
    Volume
    Single Cell Lysis Solution (Ambion single cell-to-Ct qRT-PCR Kit)
    12.75 μl
    C1 Lysis Plus Reagent (C1 Single-Cell Auto Prep Reagent Kit)
    4.35 μl
    C1 DNA Dilution Reagent (C1 Single-Cell Auto Prep Reagent Kit)
    1 μl
    *Vortex thoroughly
  4. Reverse transcription Final Mix (Total 12.4 μl)
    Components
    Volume
    Stop Solution (Ambion single cell-to-Ct qRT-PCR Kit)
    1.94 μl
    Single cell VILO RT Mix (Ambion single cell-to-Ct qRT-PCR Kit)
    5.84 μl
    Single Cell SuperScript RT (Ambion single cell-to-Ct qRT-PCR Kit)
    3.62 μl
    Loading reagent (C1 Single-Cell Auto Prep Reagent Kit)
    1 μl
    *Vortex thoroughly
  5. 0.2x Pooled Taqman Primers (Total 100 μl)
    Components
    Volume
    1 μl of each Taqman Probe (x96 probes)
    1 μl each
    C1 DNA Dilution Reagent (C1 Single-Cell Auto Prep Reagent Kit)
    4 μl
    *Vortex thoroughly
    **This reagent can be made in advance and aliquots stocked up to 6 months at -20 °C.
  6. Pre-amplification Mix (Total 60 μM)
    Components
    Volume
    Single-Cell PreAm Mix (Ambion single cell-to-Ct qRT-PCR Kit)
    12 μl
    Loading Reagent (C1 Single-Cell Auto Prep Reagent Kit)
    3 μl
    Pooled primers (The mix previously prepared)
    15 μl
    DNA-free water
    30 μl
    *Vortex thoroughly
  7. 10x Assays for single-cell qPCR
    In a qPCR 96-well plate, in each inlet add the following mixture for each of the Taqman assays.
    Components
    Volume per inlet
    Volume 10 reactions stock
    20x Taqman gene expression Assay
    2.5 μl
    25 μl
    2x Assay Loading Reagent
    2.5 μl
    25 μl
    **This reagent can be made in advance and stocked up to 6 months at -20 °C
  8. Sample pre-mix
    Components
    Volume per inlet
    Sample pre-mix for all wells
    Taqman Universal PCR Master Mix
    2.5 μl
    360 μl
    20X GE Sample Loading Reagent
    0.25 μl 36 μl

Acknowledgments

The present work was supported in part by a Grant-in-Aid for Scientific Research (C) JSPS KAKENHI Grant Number: 19K16761, Grant-in-Aid for Young Scientists (A) JSPS KAKENHI Grant Number: 17H04991 and the “Development of Diagnostic Technology for Detection of miRNA in Body Fluids” grant from the Japan Agency for Medical Research and Development (AMED).
  This protocol was adapted from Fluidigm system manufacture’s protocols (PN-100-6177 J1 and PN 68000130 E1).

Competing interests

We have no conflict of interest to declare.

References

  1. Karaiskos, N., Wahle, P., Alles, J., Boltengagen, A., Ayoub, S., Kipar, C., Kocks, C., Rajewsky, N. and Zinzen, R. P. (2017). The Drosophila embryo at single-cell transcriptome resolution. Science 358(6360): 194-199.
  2. Katsuda, T., Hosaka, K., Matsuzaki, J., Usuba, W., Prieto-Vila, M., Yamaguchi, T., Tsuchiya, A., Terai, S. and Ochiya, T. (2019). Transcriptomic dissection of hepatocyte heterogeneity: linking ploidy, zonation, and stem/progenitor cell characteristics. Cell Mol Gastroenterol Hepatol.
  3. Livak, K. J., Wills, Q. F., Tipping, A. J., Datta, K., Mittal, R., Goldson, A. J., Sexton, D. W. and Holmes, C. C. (2013). Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells. Methods 59(1): 71-79.
  4. Prieto-Vila, M., Usuba, W., Takahashi, R. U., Shimomura, I., Sasaki, H., Ochiya, T. and Yamamoto, Y. (2019). Single-cell analysis reveals a preexisting drug-resistant subpopulation in the luminal breast cancer subtype. Cancer Res 79(17): 4412-4425.
  5. Prieto-vila, M., Yamamoto, Y., Takahashi, R. Ochiya, T. (2018). Single-cell transcriptomics. In: Handbook of Single Cell Technologies. Springer, Singapore.
  6. Rato, S., Golumbeanu, M., Telenti, A. and Ciuffi, A. (2017). Exploring viral infection using single-cell sequencing. Virus Res 239: 55-68.
  7. Van den Brink, S. C., Sage, F., Vértesy, Á., Spanjaard, B., Peterson-Maduro, J., Baron, C. S., Robin, C. and van Oudenaarden, A. (2017). Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat Methods 14(10): 935-936.
  8. Wen, L. and Tang, F. (2016). Single-cell sequencing in stem cell biology. Genome Biol 17: 71.
  9. Woyke, T., Doud, D. F. R. and Schulz, F. (2017). The trajectory of microbial single-cell sequencing. Nat Methods 14(11): 1045-1054.
  10. Zheng, C., Zheng, L., Yoo, J. K., Guo, H., Zhang, Y., Guo, X., Kang, B., Hu, R., Huang, J. Y., Zhang, Q., Liu, Z., Dong, M., Hu, X., Ouyang, W., Peng, J. and Zhang, Z. (2017). Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 169(7): 1342-1356 e1316.
  11. Ziegenhain, C., Vieth, B., Parekh, S., Reinius, B., Guillaumet-Adkins, A., Smets, M., Leonhardt, H., Heyn, H., Hellmann, I. and Enard, W. (2017). Comparative Analysis of Single-Cell RNA Sequencing Methods. Mol Cell 65(4): 631-643 e634.

简介

[摘要 ] 单细胞转录组是在一个细胞中表达的信使RNA分子的集合。它变化很大,并会根据外部,物理和生化条件而变化。由于敏感性不足,大多数基因研究使用大量样品,仅提供平均基因表达。单细胞技术提供了一种强大的方法,可以更详细地了解异质群体和少数细胞。然而,因为它仍然是一个相当新的技术,标准化协议具有至b e建立。尽管单细胞qPCR受基因数量的限制,但分析起来相对简单。因此,无需使用复杂的生物信息学分析就可以使用它。如本协议所述,单细胞qPCR的主要步骤包括单细胞分离,细胞裂解液,cDNA逆转录合成,cDNA库生成的扩增以及最终的定量聚合酶链反应。

[背景 ] 的单细胞转录是一套完整的在一个细胞中表达的信使RNA(mRNA)分子。它变化很大,并会根据外部,物理和生化条件而变化。因此,这是生物异质性的来源,其中来自相同环境的细胞与其他细胞相似但不相同。

由于灵敏度不足,大多数遗传研究使用大量样本,其中有数百至数千个细胞,仅提供平均基因表达。这极大地限制了少数细胞群体的研究,这可能会带来特定的特性,例如在癌症情况下的耐药性或转移能力。单细胞技术能够在单细胞水平上分析转录组,从而揭示了异源群体的复杂性。这些技术不仅应用于癌症,而且还应用于许多细胞生物学研究中,包括成年组织,干细胞,免疫细胞等。以及微生物学和病毒学等其他领域(Wen and Tang,2016; Karaiskos 等,2017; Rato 等,2017; Woyke 等,2017; Zheng 等,2017)。

单细胞转录组方法主要有两种:mRNA测序和定量聚合酶链反应。尽管mRNA测序可以在没有先验知识的情况下研究整个转录组,但是对于该领域的新研究人员而言,操纵大量基因可能是不可行的。此外,需要高水平的生物信息学(Prieto-vila 等人,2018)。为了平衡,单细胞qPCR分析仅限于一定数量的基因,但可以作为普通qPCR分析,更易于使用。

在我们之前的研究中,我们旨在通过在乳腺癌细胞系中使用单细胞qPCR研究细胞的耐药亚群。为了这个目的,我们使用了大规模平行的单细胞扩增,这使我们能够在单细胞水平上研究96个基因,每次运行96个细胞(Prieto-Vila et al。,2019)。这是用于单细胞qPCR的最常用技术之一,其中反应在特定的96孔板中进行,其中单个样品同时被逆转录。C1 纳米流体以及Fluidigm 系统是sc - qPCR 最常用的系统。C1机器是一种流体动力细胞捕集器芯片,由通道网组成,在其中洗脱细胞,单个细胞从大小上分开。多亏了这个系统,很少发现双峰。然后将96个单细胞逃逸到96个单独的孔中。在这些孔中,使用很小的反应量即可并行进行裂解,逆转录和qPCR的预扩增(Ziegenhain 等人,2017)。接着,通过Fluidigm 系统分析96种单个基质细胞中96种基因在基质-芯片中的表达,从而进行大规模并行qPCR 。

在本协议中,我们提供了一个单细胞转录组分析方案,包括使用Fluidigm 系统执行单细胞qPCR的实验细节。

关键字:单细胞, 转录组学, 单细胞qPCR, mRNA, 微流控系统, 细胞异质性

材料和试剂


 


移液管头1000 微升/ 200 微升/ 20 微升(索伦森Bioscience公司,目录NU MBER 小号:34000/14220/15020)
低结合力离心管1.5 ml(Watson BioLab ,目录号:pk-15 c-500)
Rainin LTS 吸头10μl (Rainin ,目录号:30389228)
qPCR的96孔板(应用乙iosystems,目录号:4346907)
用于qPCR板的胶片(Applied B iosystems,目录号:4311971)
无核酸酶的水(Ambion ,目录号:AM9932)
 


用于细胞培养


细胞培养100毫米培养皿(Thermo Fisher Scientific,目录号172931)
15 ml锥形离心管(Thermo Fisher Scientific,目录号:339650)
所需的细胞系,在这种情况下,我们使用了乳腺癌细胞系MDA-MB-231和MCF7
RPMI 1640x基本介质(Thermo Fisher Scientific,目录号:C11875500BT)
胎牛血清(FBS)(Gibco,目录号:10270-106)
注意:FBS应在56℃下热灭活 °C 30分钟后使用。


抗生素/抗有丝分裂溶液(Thermo Fisher Scientific,目录号:15240062)
磷酸盐缓冲液(PBS)(DS Pharma Biomedical,目录号:DSBN200)
TrypLE Express(Thermo Fisher Scientific,目录号:12604013)
台盼蓝溶液0.4%(Gibco,货号:15250-061)
Neubauer改进的C芯片(NanoEnTek ,目录号:DHC-N01)
0.5 mol / L EDTA(Nacalai Tesque ,目录号:06894-14)
初始细胞悬液缓冲液(请参阅食谱)
 


用于单细胞分离和预扩增


C1单节自动准备IFC用于前置放大器(10-17 微米)(Fluidigm公司,目录号:100-5749)
注意:有多个通道尺寸板可调整到像元大小。有关更多信息,请参见注释。


20x基因特异性检测(Taqman )
C1单细胞自动制备试剂盒(Fluidigm ,目录号:100-5319)
Ambion 单细胞对Ct qRT -PCR试剂盒(Thermo Fisher Scientific,目录号:4458237)
哺乳动物细胞的活/死试剂盒(生命技术,目录号:L-3224)(s 食谱)
裂解混合物(请参见食谱)
反转录最终混合物(请参阅食谱)
0.2x合并Taqman 底漆(请参阅食谱)
前置放大混音(请参阅食谱)
 


对于单细胞qPCR


Fluidigm 96.96定量PCR动态阵列微流控芯片(Fluidigm ,目录号:BMK-M-96.96GT )
Taqman Universal PCR Master Mix 2x(Applied Biosystems,目录号:PN 4304437)
20x GE样品加载试剂(Fluidigm ,目录号:PN 100-7610)
2x上样试剂(Fluidigm ,目录号:PN 100-7611)
单细胞qPCR的10次检测(请参阅食谱)
样品预混(请参阅食谱)
 


设备


 


纽鲍尔室
微量移液器(P2,P10,P20,P200,P1000)
高精度多通道移液器P10(Rainin ,目录号:17013802)
细胞培养培养箱:37 °C 和5%CO 2 (Panasonic Healthcare,目录号:MCO-170AICUVH-PJ)
无尘工作台(松下,目录号:MCV-131BNF-PJ)
离心1.5 ml管(Eppendorf ,型号:5418R )
15 ml管离心机(Tomy ,型号:Ax-511 )
96孔板离心机(Sigma ,型号:4-16KS )
Vortex (科学工业公司,型号:Vortex - Genie 2 )
荧光共聚焦显微镜(Keyence ,型号:BZ-X700 )
冰箱和冰柜(任何公司的产品都可以)
C1自动准备系统(Fluidigm )
IFC 控制器HX(Fluidigm )
用于实时定量PCR的BioMarkHD 系统(Fluidigm )
 


软件


 


Fluidigm 实时PCR分析软件v.2.1.3
Fluidigm 数据收集软件v.2.1.3
电子表格
R(版本3.3.2)
 


程序


 


单细胞qPCR方案可以主要划分d INT O 4个馏分:所述第一个包括细胞培养和细胞悬液的制备。第二个和第三个主要由C1集成流体回路完成,它允许单细胞分离,细胞裂解和预扩增。最后一部分是单一实时qPCR,由BioMarkHD 系统完成。中表示的那些步骤˚F igure 1。


 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig1.jpg


图1.单细胞qPCR协议方案


 


样品制备
该协议从已经进行的细胞培养开始。


当细胞达到融合度的70%时,随时可以收集。
除去培养基,并用PBS清洗培养皿一次。
加入1毫升的TrypLE Express。
放置在37 °C 的培养皿中,并孵育3-5分钟,直到细胞分离。
在37 °C 下加入4 ml预热的完全培养基以终止反应。
小心移液以确保所有细胞完全分离,但不损坏细胞。
将细胞转移到15 ml管中。
在300 xg 4 °C下离心5分钟。
吸出培养基并加入5 ml PBS 。
分开100 微升计数细胞。
将15毫升试管在300 xg 4 °C下离心5分钟。
离心时,将分离的细胞与台盼蓝溶液按1:1比例混合。
将10μl 混合液插入Neubauer腔室。
用显微镜计数总细胞数(包括死细胞)。
除去PBS。
将细胞以300细胞/ 微升(3 x 10 5 细胞/毫升)的浓度重新悬浮在初始细胞悬液缓冲液(配方1)中,并置于冰上。
注意:建议至少10,000个细胞的数量以获得完整的捕获,我们观察到,少于该数量的细胞会留下多个通道,而IFC板中未附着细胞。


 


单细胞分离
从储备的-20 °C中取出所有必要的试剂,并在开始实验前在冰上融化30分钟。刚开始使用之前,请先将试剂保存在4 °C的温度下,然后将其放在冰上。
注意:切勿将盘子放在冰上。


卸下位于集成流体回路(IFC)板按钮处的黑色塑料。
在启动之前,请打开C1机器,因为它需要一些时间才能启动(大约3分钟)。
按照该方案吸取几种试剂:
 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig2.jpg


图2. IFC板的示意图,指示要添加不同试剂和溶液的孔。相同的数字表示相同的溶液和体积。灰色的孔表示被纸膜覆盖的芯片部分,在实验结束时,该膜将包含预先扩增的每个单细胞样品,准备在sc -qPCR中使用。


①。加入200 微升收获试剂。此孔就像一个在铁心轴上带有塑料盖的按钮。要施加试剂,请用笔尖轻轻按一下,然后滑落,将笔尖放在空间上。然后慢慢加入试剂。如图3所示。     


②。加入20 微升收获试剂。     


③。移液15μl 封闭剂。     


④。加入20 微升预压试剂。     


⑤。加入20 微升洗涤液的。     


 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本雄介811073 \ Figs jpg \ Fig3.jpg


图3. 在IFC板上应用Harvest试剂(200μl )的方案


 


将印版插入IFC机器,条形码朝外。
注意:如果板的底部脏了,机器将无法检测到它并给出错误信息。如果是这样,请用70%的乙醇擦拭底部,然后再次放置。


根据所选的芯片通道大小,运行模式STA :Prime (1782x / 1783x / 1784x)。此过程需要10到12分钟。
在灌注时,准备细胞混合物。


制备细胞混合物:30 微升细胞悬浮液和20 微升悬浮液试剂。
注意:只要保持比例,就可以制备较少量的细胞混合液。例如,6μl 和4μl 。


灌注完成后,单击弹出并取出板。
用P200移液器除去残留的C1封闭剂(孔如图2 - ③所示)。
插入细胞和活/死染色溶液,如下所示:
 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig4.jpg


图4. IFC板的示意图,指示要添加不同试剂和溶液的孔


①。加入6μl 细胞混合物。     


注意:请勿涡旋细胞。加入前用移液器吸取。


②。加入20 μl的活/死染色溶液(配方2)的。     


 


将IFC板放入C1系统。
根据所选的芯片通道大小,选择“ STA:Cell Load&Stain (1782x / 1783x / 1784x)” 。这需要30-65分钟,具体取决于板的细胞大小。
同时,准备Lysis Mix,RT Mix和PreAmp Mix(配方3、4和5)。
加载完成后,从C1中取出板并用Keyence显微镜拍摄细胞照片。使用10倍的相衬,可以轻松地在板的中央部分观察到通道和荧光染色的细胞,在图4中用灰色显示。还可以观察到每个孔的数量。
记下有死细胞的通道,空通道和有一个以上要丢弃的细胞的通道(图5)。
 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig5.jpg


图5.活/死染色示例。绿色细胞是活细胞,而阅读细胞则死了,或者是高度受损的细胞。此外,右室显示双峰,左室显示透明单细胞。


 


细胞裂解,逆转录和预扩增。
按照图6中的方案添加试剂:
 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig6.jpg


图6. IFC板的示意图,指示要添加不同试剂和溶液的孔


①。加入180μl 的Harvest试剂。小心地从一侧移至另一侧,以确保试剂覆盖所有表面。     


②。添加7 微升裂解最终修复。     


③。添加7 微升RT最后的混音的。     


④。加入24μl 的预扩增混合物(配方6)。     


 


将板放入C1系统。
选择STA:PreAmp (1782x / 1783x / 1784x)– 根据所选的芯片通道大小,然后选择结束时间。我们通常将其放置一夜,并于第二天早晨收集它以进行实验。该过程本身大约需要5-6小时。
STA:前置放大器过程完成后,选择EJECT并卸下IFC板。
使用新的PCR 96孔板,在每个孔中添加25μlC1 稀释试剂。
取出纸张,然后保护IFC板上的输出孔(这些盖板在图2、4和6中用灰色表示)。
使用八通道微量移液器(Rainin ),将样品转移到96孔板上。
注意:样品量应接近3μl ,但要确保将微量移液器设置为4μl 。


使用此特定顺序转移样品,如图7所示。
注意:使用8通道微量移液器,并考虑到IFC板每条泳道有16孔,因此它的大小可确定其他每孔的大小。


 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig7.jpg


图7.说明从IFC板转移到PCR 96孔板的顺序的方案


 


涡旋96孔板并向下旋转。此时,样品可以在-20 °C 下冷冻,并在使用前保存数周,在这种情况下,请使用PCR板膜将板密封。
 


单细胞qPCR
从储备的-20 °C中取出所有必要的试剂,并在开始实验前在冰上融化30分钟。刚开始使用之前,请先将试剂保存在4 °C的温度下,然后将其放在冰上。
注意:切勿将盘子放在冰上。


打开标准96孔热循环仪,因为它需要一些时间才能启动。
去除96.96芯片底部的蓝色保护膜。
使用工具包中的注射器,将控制管线流体注入两个大的中央孔中,并在十字之间压紧(可以看到十字,在图8中用箭头标记)。
注意:机油容易泄漏,因此在管理注射器时要小心。


 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig8.jpg


图8. 96.96 定量PCR动态阵列微流控芯片的示意图。箭头显示应在其中施加管线流体的空间。


 


插入板(条形码朝外)。
选择选项Prime(136x)。此过程大约需要20分钟。
同时,准备预混样品(配方8)。
在新的PCR 96孔板中,在每个孔中添加3.3μl 样品预混物。
添加2.7 微升cDNA样品的用多通道微量(步骤C9获得的样品)制备6的总体积微升在每个入口。
用PCR板膜密封。
准备Taqman 分析混合物(配方7)并用PCR板膜密封板。
将两个板涡旋10秒钟,然后旋转。
灌注完成后,收集芯片。
转移样品和Taqman 测定。按照以下顺序(图9和10):
 


 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig9.jpg


图9. 96.96芯片的示意图,以黄色显示插入Taqman 分析的进样口,以绿色显示插入样品预混物的进样口


①。加入5 微升的的Taqman 测定法在黄色入口,使用以下顺序(图10A),以从传送的qPCR 96孔板到IFC芯片。     


②。在绿色进样口中以相同顺序(图10A)添加5μl 样品预混物。     


笔记:


入口壁不是水平的,而是具有对角线。确保更好的应用;最好的选择是将尖端倾斜插入,如图10B所示。
移液时避免气泡。但是,如果有泡沫,但他们移动,意味着它们在表面上,和TH EN 它们不会干扰分析。如果气泡不动,用微量移液器吸头除去气泡。
 


D:\ Reformatting \ 2020-2-7 \ 1902855--1329山本佑介811073 \ Figs jpg \ Fig10.jpg


图10. 将样品转移到96.96芯片的订购方案和方法。A.说明从PCR板到96.96芯片的孔转移顺序的方案。B. 96.96个进样口中的样品应用方案。


 


将芯片插入IFC Controller HX,然后运行脚本Load Mix(136x)。
在运行“加载”脚本时,打开连接到BioMarkHD 系统的计算机以进行实时qPCR。
运行软件Biomark 数据收集。
双击打开选项“灯”以启动。此过程大约需要20-30分钟。      


加载完成后,将芯片带入BioMarkHD 系统并按照以下步骤操作:
单击“开始新运行”。
条形码朝外插入芯片。
选择:加载


 下一个


选择96.96芯片的设置。
选择名称和要将文档保存到的文件夹。
                             下一个


应用类型:单探头。
探针:FAM_MGB
 下一个


通讯协定:GE 96x96标准v1。
                             下一个


开始运行。
完成后,请卸下盖板,然后关闭软件并关闭计算机。
 


数据分析


 


数据分析程序参考我们之前的报告(Prieto-Vila et al。,2019)。


取出死/损坏的细胞
运行qPCR后,使用Biomark 软件收集要分析的原始数据。
使用设置线性(导数)和用户(检测器)设置为每个基因生成Ct值。
删除所有置信度小于0.2的值。
将数据导出到excel文件。
使用在步骤B14中从实验方案中获得的数据(活/死染色图片),去除所有染成红色(死)的细胞,一个或多个细胞的腔室,或不去除任何细胞。
删除管家基因的Ct值低于16的细胞。在我们的案例中,我们同时使用了BACTIN和GAPDH。
 


数据标准化
规范化可以通过两个系统进行:


为了消除分析之间的差异,可以使用管家基因进行归一化。在我们的案例中,我们利用了GAPDH。:归一化是通过下式计算2 - ΔΔ CT 。
在单细胞分析中另一种广泛使用的方法是通过log 2 Ex 进行归一化,该表达在log base 2中高于背景的转录水平(Livak 等,2013)。在我们的案例中,我们将LOD(检测限)设置为26,因为它是每个基因的最低可检测Ct值的平均值。这可以通过下式计算:日志2 用Ex = LOD(限位中检测)Ç q - c ^ q [基因] 。
不Ë 小号:


两种规范化都可以使用Excel完成。
有关包含示例的更详细的协议,请参阅本手册:“单一:分析工具集”(P / N 100-5066)https://www.fluidigm.com/binaries/content/assets/fluidigm/singular-analysis- toolset-3.5 / singular-analysis-toolset-3.5-userguide.pdf。
 


可以对单细胞qPCR进行多种统计分析
单细胞最常见的统计分析可能是t-SNE图,热图和ViolinPlot 。


  对于Heatmap,我们使用了软件Partek Genomics Suite Software。t-SNE和Violin Plot都是由免费软件R使用“ ggplot2”软件包完成的。


 


笔记


 


对于单细胞分析,认为在物理上也是不可能的,而不是重复,这对于增加样品数量更为重要。因此,我们将每个样本运行了两次。每个样品分析总共192个单个细胞。
三种尺寸的C1 IFC板可将通道调整为不同大小的单元。在选择板的尺寸之前,我们通过取下分离后的细胞的照片并通过Keyence软件测量尺寸来计算细胞的大小。可用的尺寸为:S(5-10 微米),M(10-17 微米),L(17-25 微米)。作为参考,我们将M大小用于MDA-MM-231和MCF7单元;并且我们已经在L尺寸平板中运行了肝细胞(Katsuda 等,2019)。
最重要的步骤之一是单细胞隔离。在此过程中,细胞是从其本地环境中分离出来的,因此,保持这些细胞存活并处于良好状态以进行适当的进一步分析是必不可少的(Van Den Brink 等人,2017)。请仔细管理它们。
该协议是根据Fluidigm 系统制造商的协议(PN-100-6177 J1和PN 68000130 E1)完成的,可以在以下网址找到:https ://www.fluidigm.com/binaries/content/documents/fluidigm/resources/ c1-taqman-pr-100%E2%80%906117 / c1-taqman-pr-100%E2%80%906117 / fluidigm%3Afile   和https://www.fluidigm.com/binaries/content/documents/fluidigm/ resources / 96.96-ge-taqman%E2%80%90std-qr-68000130 / 96.96-ge-taqman%E2%80%90std-qr-68000130 / fluidigm%3Afile。
 


 


 


菜谱


 


注意:所有配方,除非另有说明,都是一次运行所需的试剂。


初始细胞悬液缓冲液
组件数量             


EDTA(5M)0.1毫升             


FBS 50 微升             


PBS 10毫升             


*该溶液可多次使用,应保存在4 °C下。


LIVE / DEAD细胞染色溶液(总计1253.13 微升)
组件数量             


细胞洗涤缓冲液(C1单细胞自动制备试剂盒)1250 微升             


乙锭同型二聚体-1(活/死盒)2.5 微升             


钙黄绿素AM(活/死盒)0.63 微升             


裂解混合物(总计18.1 微升)
组件数量             


单细胞裂解液(Ambion公司单细胞-的Ct 的qRT -PCR试剂盒)12.75 微升             


C1裂解Plus试剂(C1单细胞自动制备试剂盒)4.35 微升             


C1 DNA稀释试剂(C1单细胞自动制备试剂盒)1 微升             


*彻底涡旋


逆转录最终混合物(总计12.4 微升)
组件数量             


终止溶液(Ambion公司单细胞-的Ct 的qRT -PCR试剂盒)1.94 微升             


单细胞RT VILO混合物(Ambion公司单细胞-的Ct 的qRT -PCR试剂盒)5.84 微升             


单细胞的SuperScript RT(Ambion公司单细胞-的Ct 的qRT -PCR试剂盒)3.62 微升             


装载试剂(C1单细胞自动制备试剂盒)1 微升             


*彻底涡旋


0.2x合并Taqman 引物(总计100μl )
组件数量             


1 微升的每个的Taqman 探针(X96探针)1 微升每             


C1 DNA稀释试剂(C1单细胞自动制备试剂盒)4 微升             


*彻底涡旋


**该试剂可预先制备,等分试样可在-20 °C下储存长达6个月。


前置扩增混合液(总计60μM )
组件数量             


单细胞PreAm 混合物(Ambion公司单细胞-的Ct 的qRT -PCR试剂盒)12 微升             


样试剂(C1单细胞自动制备试剂盒)3 微升             


合并的引物(预先制备的混合物)15 微升             


DNA-游离水30 微升             


*彻底涡旋


单细胞qPCR的10次检测
在qPCR 96孔板中,在每个入口中为每个Taqman 分析添加以下混合物。


组件每个入口的体积体积10个反应液                           


20倍的Taqman 基因表达测定法2.5 微升25 微升                           


2X测定样试剂2.5 微升25 微升                           


**该试剂可以预先制备,并且可以在-20 °C下保存6个月


样品预混
每个进样口的组分体积所有孔的样品预混合                           


的Taqman 通用PCR主混合物2.5 微升360 微升                           


20X GE上样试剂  0.25 微升36 微升                           


 


致谢


 


这项工作得到了科学研究资助(C)JSPS KAKENHI资助号:19K16761,青年科学家资助(A)JSPS KAKENHI资助号:17H04991和“诊断学的发展”的部分支持。日本医学研究与发展局(AMED)授予的“检测体液中miRNA的技术”。


  该协议改编自Fluidigm 系统制造商的协议(PN-100-6177 J1和PN 68000130 E1)。


 


利益争夺


 


我们没有利益冲突要声明。


 


参考文献


 


Karaiskos ,N.,Wahle ,P.,Alles ,J.,Boltengagen ,A.,Ayoub,S.,Kipar ,C.,Kocks ,C.,Rajewsky ,N.和Zinzen ,RP(2017)。在果蝇胚胎,在单细胞转录组的分辨率。科学358(6360):194-199。
Katsuda,T.,保坂,K.,松崎,J.,Usuba ,W.,Prieto的维拉,M.,山口,T.,土屋,A.,特莱,S。和Ochiya,T。(2019)。肝细胞异质性的转录组解剖:连接倍性,分区和干/祖细胞特征。细胞分子胃肠激素肝素。
Livak ,KJ,Wills,QF,Tipping,AJ,Datta,K.,Mittal,R.,Goldson,AJ,Sexton,DW and Holmes,CC(2013)。qPCR基因表达谱分析方法应用于1440个淋巴母细胞单细胞。方法59(1):71-79。
M.Prieto -Vila,W.Usuba ,RU,高桥,I.Shimomura,H.Sasaki,T.Ochiya和Y.Yamamoto(2019)。单细胞分析揭示了管腔型乳腺癌亚型中先前存在的耐药性亚群。癌症研究79(17):4412-4425。
Prieto- vila ,M.,Yamamoto,Y.,Takahashi,R.Ochiya,T.(2018)。单细胞转录组学。于:单细胞技术手册。新加坡施普林格。
Rato ,S.,Golumbeanu ,M.,Telenti ,A.和Ciuffi ,A.(2017年)。使用单细胞测序探索病毒感染。病毒Res 239:55-68。
V 的巢穴Brink的,SC,鼠尾草,F.,V é rtesy ,Á 。,Spanjaard ,B.,彼得森-马杜罗,J.,男爵,CS,罗宾,C.和van Oudenaarden ,A。(2017)。单细胞测序揭示了在组织亚群中解离诱导的基因表达。Nat Methods 14(10):935-936。
Wen L.和Tang F.(2016)。干细胞生物学中的单细胞测序。Genome Biol 17:71 。
Woyke ,T.,Doud ,DFR和Schulz,F.(2017)。微生物单细胞测序的轨迹。Nat Methods 14(11):1045-1054。
Zheng C.,Zheng L.,Yoo ,JK,Guo,H.,Zhang,Y.,Guo,X.,Kang,B.,Hu,R.,Huang,JY,Zhang,Q.,Liu, Z.,Dong,M.,Hu,X.,Ouyang,W.,Peng,J.和Zhang,Z.(2017)。通过单细胞测序揭示了肝癌中浸润性T细胞的景观。Cell 169(7):1342-1356 e1316。
齐根海恩,C.,Vieth ,B.,帕雷克,S.,Reinius ,B.,Guillaumet -Adkins,A.,Smets ,M.,莱茵哈特,H.,海恩,H.,海尔曼,I。和Enard ,W (2017)。单细胞RNA测序方法的比较分析。Mol Cell 65(4):631-643 e634。
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引用:Prieto-Vila, M., Ochiya, T. and Yamamoto, Y. (2020). Single-cell qPCR Assay with Massively Parallel Microfluidic System. Bio-protocol 10(6): e3563. DOI: 10.21769/BioProtoc.3563.
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