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Apr 2022

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Gene Expression Analysis in Stem Cell-derived Cortical Neuronal Cultures Using Multi-well SYBR Green Quantitative PCR Arrays
使用多孔 SYBR Green 定量 PCR 阵列在干细胞衍生的皮质神经元培养物中进行基因表达分析   

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Abstract

To optimize differentiation protocols for stem cell-based in vitro modeling applications, it is essential to assess the change in gene expression during the differentiation process. This allows controlling its differentiation efficiency into the target cell types. While RNA transcriptomics provides detail at a larger scale, timing and cost are prohibitive to include such analyses in the optimization process. In contrast, expression analysis of individual genes is cumbersome and lengthy.


Here, we developed a versatile and cost-efficient SYBR Green array of 27 markers along with two housekeeping genes to quickly screen for differentiation efficiency of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons. We first identified relevant pluripotency, neuroprogenitor, and neuronal markers for the array by literature search, and designed primers with a product size of 80-120 bp length, an annealing temperature of 60°C, and minimal predicted secondary structures. We spotted combined forward and reverse primers on 96-well plates and dried them out overnight. These plates can be prepared in advance in batches and stored at room temperature until use. Next, we added the SYBR Green master mix and complementary DNA (cDNA) to the plate in triplicates, ran quantitative PCR (qPCR) on a Quantstudio 6 Flex, and analyzed results with QuantStudio software.


We compared the expression of genes for pluripotency, neuroprogenitor cells, cortical neurons, and synaptic markers in a 96-well format at four different time points during the cortical differentiation. We found a sharp reduction of pluripotency genes within the first three days of pre-differentiation and a steady increase of neuronal markers and synaptic markers over time. In summary, we built a gene expression array that is customizable, fast, medium-throughput, and cost-efficient, ideally suited for optimization of differentiation protocols for stem cell-based in vitro modeling.


Keywords: Human iPSCs (人类 iPSC), Induced pluripotent stem cells (诱导多能干细胞), Cortical neurons (皮质神经元), Neuronal differentiation (神经元分化), SYBR Green (SYBR Green), Quantitative PCR (定量 PCR), Multi-well qPCR (多孔 qPCR), Primer design (引物设计)

Background

The real-time quantitative PCR (qPCR) technique detects amplification of target nucleic acid sequences, and it is considered sensitive, reproducible, and specific (Arya et al., 2005).


Here, we use multi-well qPCR assays to amplify multiple genes with SYBR Green technology (Arikawa et al., 2011). SYBR Green is a DNA binding dye that binds non-specifically to double-stranded DNA (dsDNA) (Boone et al., 2015).


Based on the RT2 ProfilerTM array (Arikawa et al., 2011), we designed a multi-well SYBR Green qPCR panel to analyze the expression of genes involved in the differentiation of cortical neurons from the human iPSCs by forced expression of Neurogenin 2 (Ngn2), which is a neuronal transcription factor supporting neuronal differentiation of human embryonic stem cells or iPSCs into cortical-like neurons (Zhang et al., 2013). Here, we used a human iPSC line with a doxycycline-inducible mouse Ngn2 transgene engineered into a safe harbor locus (Wang et al., 2017). We collected cells at different time points (iPSCs, Day 0 pre-neurons, Day 15, and Day 30 cortical neurons) and analyzed genes that mark pluripotent stem cells, intermediate neuroprogenitors, and mature cortical neurons on a single 96-well plate. The amplicons/ primers were designed in the range of 80-120 bp, and the Tm was between 63°C and 66°C. The in-house preparation of the multi-well SYBR Green qPCR assay allows tailoring primers to specific experiments and assay modification as needed. This multi-well SYBR Green qPCR assay can be used for the quantitative analysis of any set of genes of interest. Different RT2 profiler arrays for pathway analysis are commercially available; however, none of them are optimized to follow the maturation of cortical neurons derived from iPSCs. We show that the multi-well SYBR Green qPCR is easily adaptable for customization in the laboratory. It is an economic platform and ideally suited for the optimization of differentiation protocols for in vitro stem cell modeling.

Materials and Reagents

  1. RNA extraction

    1. Homogenizer spin column (Thermo Fisher Scientific, Life Technologies, catalog number: 12183-026)

    2. 2-mercaptoethanol (Sigma-Aldrich, Aldrich Chemistry, catalog number: M2650)

    3. DNase I, amplification grade (Thermo Fisher Scientific, Invitrogen, catalog number: 18068015)

    4. Ethanol, molecular grade (Thermo Fisher Scientific, catalog number: BP2818500)

    5. PureLinkR RNA mini kit (Thermo Fisher Scientific, Life Technologies, catalog number: 12183025)

    6. RNase away (Thermo Fisher Scientific, Life Technologies, catalog number: 10328011)


  2. cDNA Reverse Transcription

    1. MicroAmp 8-tube strip (Thermo Fisher Scientific, Applied Biosciences, catalog number: A30589)

    2. High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Invitrogen, catalog number: 4368814)


  3. Multi-well PCR reaction

    1. MicroAmp Optical 96-well reaction plate (Thermo Fisher Scientific, Applied Biosystems, catalog number: N8010560)

    2. MicroAmp Optical adhesive film (Thermo Fisher Scientific, Applied Biosystems, catalog number: 4311971)

    3. Nuclease-free water (US Biological LifeSciences, catalog number: W0900)

    4. PowerUpTM SYBRTM Green master mix (Thermo Fisher Scientific, Applied Biosystems, catalog number: A25780)


  4. Consumables

    1. 1.5 mL and 0.6 mL RNase-free microcentrifuge tubes

    2. RNase-free filter pipette tips (P1000, P200, P20, and P2)


  5. Others

    1. 10× PBS, molecular grade (Fisher Scientific, catalog number: J75889K2

    2. Accutase (Thermo Fisher Scientific, catalog number: NC9464543)

    3. Bucket with wet ice

    4. Personal protective equipment (gloves, lab coat, goggles)

Equipment

  1. Revco Ultima II ultra low temperature -86°C freezer (Thermo Scientific, catalog number: ULT2586-9)

  2. Microcentrifuge 5415C (Eppendorf, catalog number: M7282)

  3. Refrigerated centrifuge (Beckman Coulter, catalog number: GS6 Allegra)

  4. Mini centrifuge (Fisher Scientific, catalog number: 05-090-100)

  5. Water bath (Thermo Fisher Scientific, Cole Parmer, catalog number: TSGP20)

  6. MiniAmpTM thermal cycler (Applied Biosystems, Thermo Fisher Scientific, catalog number: A37834)

  7. NanoDrop spectrophotometer (Thermo Fisher Scientific, catalog number: 13-400-525)

  8. QuantStudio 6 Flex (Applied Biosystems, Thermo Fisher Scientific, catalog number: 4485691)

  9. Optional: PlateR visual pipetting aid tablet (Biosistemika, catalog number: P-10)

Software

  1. Beacon Designer (Premier Biosoft, http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1)

  2. In silico PCR prediction (UCSC Genome Browser, https://genome.ucsc.edu/cgi-bin/hgPcr)

  3. Primer3 software (Whitehead Institute for Biomedical Research, Steve Rozen, Maido Remm, Triinu Koressaar, and Helen Skaletsky, https://bioinfo.ut.ee/primer3-0.4.0/)

  4. QuantStudio Flex 6-v1.7.1 (Applied Biosystems, ThermoFisher Scientific, https://www.thermofisher.com/us/en/home/global/forms/life-science/quantstudio-6-7-flex-software.html)

  5. UNAFold (Integrated DNA technology, https://www.idtdna.com/UNAFold). Create an account to use the software

Procedure

  1. Ngn2-guided cortical neuron sample collection

    The iPSCs seeded at 1.5 × 105 cells, were differentiated in a 12-well plate (~40,000 cells per cm2) according to the protocol of Wang et al. (2017) using an Ngn2-inducible cell line. The media was changed every other day. As shown in Figure 1, cells were collected at day -3 (3 days before doxycycline induction), day 0, day 15, and day 30 for iPSCs, pre-neurons, day 15, and day 30 cortical neurons, respectively, for the illustration of this SYBR Green multi-well array. A confluent well of a 12-well plate of iPSCs yields 3 × 106–4 × 106 cells, and the RNA yield will be approximately 8–10 μg total RNA. For pre-neurons, the RNA yield ranges between 5-6 μg from 2 × 106–3 × 106 cells, and for neurons (days 15 and 30), RNA yield ranges between 1–3 μg from 1 × 106–2 × 106 cells.



    Figure 1. Timeline for the Ngn2-guided cortical neuronal differentiation and collection of samples for RNA extraction. Scale bar: 100 μm.


    1. To dissociate the cells for sample collection, aspirate the old media, directly add 400 μL of Accutase per well of a 12-well plate, and incubate the cells at 37°C for 3–5 min to lift them off.

    2. Add 400 μL of 1× cold PBS to Accutase, pipette the cell solution up and down to dissociate, and transfer the cell suspension to a 1.5 mL Eppendorf tube. Wash the well with additional 400 μL of 1× cold PBS to collect the remaining cells and add to the same Eppendorf tube.

    3. Centrifuge the cell suspension at 15,000 × g at 4°C for 5 min.

    4. Aspirate and discard the supernatant.

    5. Wash the cell pellet with 1 mL of 1× cold PBS by pipetting up and down several times, and centrifuge again at 15,000 × g at 4°C for 5 min.

    6. Aspirate the supernatant while keeping it on wet ice.

    7. Continue with RNA extraction or store the cell pellet at -80°C.


  2. RNA extraction and purification (PureLinkR RNA Mini Kit, Thermo Fisher, Life Technologies, 12183025)

    Preparation:

    1. Clean the bench with 70% ethanol and RNase Away.

    2. Use RNase-free barrier filter tips for RNA extraction.

    3. Before using Wash Buffer II for the first time:

      1. Add 60 mL of 96-100% ethanol directly to the Wash buffer bottle.

      2. Check the box on the Wash Buffer II label to indicate that ethanol was added.

      3. Store Wash Buffer II with ethanol at room temperature.

    4. Prepare fresh Lysis Buffer containing 1% 2-mercaptoethanol for each purification. Under the chemical hood, add 3 μL of 2-mercaptoethanol per 300 μL of Lysis Buffer.


    Procedure:

    1. Take the cell pellet from the -80°C freezer and place it on ice (perform subsequent steps at room temperature).

    2. Add 300 μL of Lysis Buffer to the cell pellet.

    3. Vortex for 10 s at high speed until the cell pellet is thoroughly mixed.

    4. Transfer the lysate to a homogenizer spin column inserted in an RNase-free Eppendorf tube and centrifuge at 12,000 × g for 2 min. Remove and discard the homogenizer cartridge after centrifugation.

    5. Add 300 μL of 70% ethanol to the cell homogenate.

    6. Vortex to mix thoroughly and to disperse any visible precipitate that may form after adding ethanol.

    7. Transfer up to 700 μL of the sample to a spin cartridge (with a collection tube).

    8. Centrifuge at 12,000 × g for 15 s at room temperature. Discard the flow-through and reinsert the spin cartridge into the same collection tube.

    9. Add 700 μL of Wash Buffer I to the spin cartridge. Centrifuge at 12,000 × g for 15 s at room temperature. Discard the flow-through and the collection tube. Place the spin cartridge into a new collection tube.

    10. Add 500 μL of Wash Buffer II (supplemented with ethanol) to the spin cartridge.

    11. Centrifuge at 12,000 × g for 15 s at room temperature. Discard the flow-through and reinsert the spin cartridge into the same collection tube.

    12. Repeat steps 10-11 once.

    13. Centrifuge the spin cartridge at 12,000 × g for 2 min to dry the membrane containing the RNA.

    14. Discard the collection tube and insert the spin cartridge into a new recovery tube.

    15. Add 30 μL of RNase-free water to the center of the spin cartridge.

    16. Incubate at room temperature for 1 min.

    17. Centrifuge the spin cartridge for 2 min at ≥12,000 × g at room temperature to elute the RNA from the membrane into the recovery tube.

    18. Add the eluted RNA sample to the same spin cartridge again and repeat steps 16–17 to increase the yield of the RNA sample.

    19. The concentration of the eluted RNA is determined using a NanoDrop spectrophotometer.

    20. The concentration of RNA varies between cell types as mentioned in Figure 2.

    21. Critical step! Aliquot the RNA into RNase-free tubes, each having 1 μg of RNA, to avoid freeze-thaw cycles and degradation of RNA. The aliquot volume for 1 μg of RNA is directly used for the DNase I treatment.

      Note: The aliquots are treated with DNase I before storage.

    22. Proceed with DNase I treatment directly after RNA purification or freeze aliquots at -80°C.

    23. It is absolutely critical to aliquot RNA and cDNA samples to avoid freeze-thaw cycles and avoid degradation of the sample.



    Figure 2. Workflow and calculations for the different cell types through the cortical neuron differentiation with stopping points and storage.


  3. DNase I Treatment (Thermo Fisher Scientific, Invitrogen, catalog number: 18068015)

    1. Combine the items shown in Table 1 in RNase-free tubes:


      Table 1. Reaction mix for DNase I treatment of eluted RNA

      Component Volume
      RNA (1 μg) up to 8 μL
      10× DNase I Buffer 1 μL
      RNase-free Water add up to 8 μL
      DNase I, Amplification Grade 1 μL
      Final Volume 10 μL


    2. Mix and incubate for 15 min at room temperature.

    3. Heat-inactivate the DNase I by adding 1 μL of 25mM EDTA to the DNase I treated RNA sample and place it in a water bath at 65°C for 3 min.

    4. Once the RNA samples are DNase I heat-inactivated, place them on ice.

    5. Proceed with the reverse transcription or store aliquots at -80°C.


  4. cDNA Reverse Transcription (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher, Invitrogen, catalog number: 4368814)

    The extracted RNA is converted to cDNA for qPCR amplification. As a control, prepare a reverse transcription control with no addition of the reverse transcriptase in the reaction mix.

    The reaction mixture (RT mix) is prepared according to Table 2. The 1 μg RNA (10 μL volume) from the previous step is mixed with the reaction mix (Table 2) for a final volume of 20 μL for the cDNA reaction.


    Table 2. Reaction mix for the cDNA reverse transcription reaction.

    Component Volume/reaction (μL)
    10× RT Buffer 2.0
    25× dNTP Mix (100mM) 0.8
    10× RT Random Primers 2.0
    MultiscribeTM Reverse Transcriptase 1.0
    RNase Inhibitor 1.0
    Nuclease-free water 3.2
    Total volume per reaction mix (RT mix) 10.0


    1. Preparation of cDNA RT reaction:

      1. The RT mix is prepared, mixed gently, and placed on ice.

      2. Pipette 10 µL RT master mix into MicroAmp 8-tube strip.

      3. Add 10 µL of DNase I treated RNA sample (1 μg) to the reaction mix and mix by pipetting up and down several times.

        Notes:

        1. One tube has to be prepared containing the RNA input but not the MultiscribeTM Reverse Transcriptase. This is used as reverse transcriptase control (RTC), which can confirm that no genomic DNA is amplified.

        2. Do not introduce bubbles while pipetting.

      4. The tubes are sealed and centrifuged to spin down the contents and eliminate air bubbles.

      5. Place the tubes on ice and load them into the MiniAmpTM Thermal Cycler.

    2. Reverse transcription thermal cycling conditions (Table 3):

      1. The reaction volume is set to 20 µL.

      2. Load the reaction tubes into the thermal cycler.

      3. The thermal cycler program is set to RUN.

      4. Once the cycle is complete, add 80 μL of nuclease-free water to the 20 μL cDNA for a working concentration of 10 ng/μL, store the cDNA at -80°C.

      5. Critical step! Aliquot the sample to avoid freeze-thaw cycles and degradation of cDNA.


        Table 3. Thermal cycler conditions of cDNA reaction.

        Step 1 Step 2 Step 3 Step 4
        Temperature (°C) 25 37 85 4
        Time (min) 10 120 5


  5. Primer Design

    We designed SYBR Green primers using an established protocol (Thornton and Basu, 2011). The primer parameters of this section are: Product size: 80–120 bp, product melting temperature (Tm): 63–66°C, secondary structure ΔG: not more than -3.5 and the GC percentage: 35–80%, with optimal at 65%. The primers were designed in an intron-spanning fashion, so that the forward and the reverse primer were not placed in the same exon. This can avoid contamination from genomic DNA.


    1. Step 1: To obtain the sequence of the gene of interest in FASTA format from the National Center for Biotechnology Information (NCBI) website: http://www.ncbi.nlm.nih.gov.

      1. Select the Nucleotide option from the dropdown menu in search “All Databases”.

      2. Enter the gene name or the sequence ID of interest in the search box and click on Search.

      3. Since the cortical neurons are differentiated from the human iPSCs, either mention homo sapiens along with the gene name in the search bar, or select homo sapiens from the species filter on the top left of the webpage after clicking on search.

      4. Click on the RefSeq transcripts and select the FASTA option, then click apply.

    2. Step 2: After obtaining the FASTA sequence for the selected gene, design the primers using Primer3 software: https://bioinfo.ut.ee/primer3-0.4.0/ (SantaLucia, 1998), Figure 3.

      Note: The explanation for the default values is given in the webpage: https://bioinfo.ut.ee/primer3-0.4.0/input-help.htm.



      Figure 3. Screenshot of Primer3 webpage indicating the parameters and conditions for designing primers of the SYNAPSIN1 (SYN1) gene.


      1. Copy and paste the FASTA format sequence of the gene interest into the box provided on the Primer3 primer design page.

      2. Pick left primer: This option was left blank for the software to pick the primers.

      3. Pick hybridization probe: This option was left blank (not required with this experiment).

      4. Pick right primer: If this option is left blank, the Primer3 program will choose the right primer.

      5. Sequence Id (Name of the gene): This is to identify the primers for the sequence. A proper name is set corresponding to the sequence.

      6. Targets (region of the sequence of the gene of interest): After looking into the CDS and exon regions (shown in Figure 4) the specific nucleotide position in the sequence is entered against the target, following with the number of nucleotides along to be flanked for the primers to design surrounding that particular region. An example, on how to enter the target regionis is shown in Figure 4.



        Figure 4. Image of NCBI webpage indicating the position of exons


      7. Primer Tm: This is the temperature at which 50% of the primer is hybridized to the DNA template. For this experiment, all primers are designed in the range of 63–66°C Tm.

      8. Maximum Tm difference: Enter the value as 2.

      9. Table of Thermodynamic parameters: Primer3 uses these formulas to calculate the melting temperature. Set the method to SantaLucia (1998).

      10. Product Tm: This is the temperature at which 50% of the amplicon is ssDNA. Set the optimal value to 50.

      11. Primer GC: This is the minimum and maximum percentage of guanine and cytosine (GC) allowed. The GC content of primers is used to determine the melting temperature of the primer, which can be used to predict the annealing temperature. Set the values to Minimum: 35, Optimum: 65, Maximum: 80.

      12. Max Self-complementary: Primers should not be self-complementary or complementary to each other. Primers that are self-complementary form self-dimers or hairpin structures. Enter the value as 4.

      13. Max 3’ Self-complementary: As polymerases add bases at the 3’ end of the oligonucleotide, the 3’ ends of primers should not be complementary to each other, as primer dimers will occur. Enter the value as 3.

      14. Max #N: This is the maximum number of unknown bases which Primer3 could consider for designing primers. Set value at 0.

      15. Max Poly-X: The maximum number of mononucleotides repeats to allow in the primer. Long mononucleotide repeats can promote mispriming. Enter the value as 3.

      16. Inside target penalty and outside target penalty: Used if the primer needs to be designed to overlap a region. Leave as default.

      17. First Base Index: This parameter tells Primer3 which programming index type the first base in the input sequence is. Leave as default.

      18. GC Clamp: Defines the specific numbers of Gs and Cs at the 3’ end of both the left and right primers. Leave the value as 0.

      19. Conc. of monovalent cations: This is the millimolar concentration of KCl salt in the PCR. Enter the value as 50 µM.

      Note: According to the SantaLucia (1998), the concentration for monovalent cations is assumed at 50 μM and at 3.5 mM for divalent cations. Other literature suggests a range for monovalent cations between 20 to 100 μM and divalent cations between 1.5 to 5 mM.


      1. Salt correction formula: Factors such as ΔG and Tm affect PCR performance and alter the efficiency of primer pairs. The SantaLucia (1998) salt formula is preferred by Primer3. This formula is designed to accommodate the salt correction independent of sequence but dependent on oligonucleotide length.
      2. Conc. of divalent cations: This is the concentration of divalent salts present in the PCR mix. Set value at 3.5 mM.

      3. Conc. of dNTPs: A dNTP concentration of 200 µM is usually recommended for Taq polymerase to function efficiently in a conventional PCR. Some SYBR Green master mixes come with Taq, KCl, MgCl2, and dNTP. These mixes tested in laboratories give maximum performance. Enter the value as 0.20 mM

      4. Annealing Oligo Concentration: Used to calculate the oligo melting temperature, this is the nanomolar concentration of annealing oligos in the PCR. Leave at default.

      5. Objective function penalty weights for Primers: The penalty weights section allows Primer3 users to modify the criteria that Primer3 uses to select the best sets of primers.

        Objective Function Penalty Weights for Primers:

        • Tm Lt = 1; Gt = 1

        • Size Lt = 1; Gt =1

        • Self complementary = 3

        • 3’ Self complementary = 3

        • #N’s = 2

        • All other values = 0

        Objective Function Penalty Weights for Primer Pairs:

        • Product Tm: Lt = 1; Gt = 1

        • Tm difference = 2

        • Any complementary = 3

        • 3’ complementary = 3

        • Primer Penalty weight = 1

        • All other values = 0

    3. Analyzing primers: Once all the Primer3 parameters are set as mentioned above, Primer3 will design primer pair options.

      1. Once all options are entered, press ‘Pick Primers’.

      2. The sequence will be displayed under the primers with the details on the primers generated, and the location of the primers within the sequence is indicated by >>>>>>> for the forward primer and <<<<<< for the reverse primer. The first primer that has the correct product length and Tm is analyzed for the secondary structure using Beacon DesignerTM free edition. Select the primer pair based on the 3′ value, which should not be more than 3.00. The 3′ value is the measurement of primer dimers formed within the primer pair (Figure 5).



      Figure 5. Image of Primer3 output indicating the 3′ value, for the primer dimers in SYN1


    4. Use Beacon DesignerTM free edition to check for primer secondary structures. The acceptable ΔG value for the primers should not be more than -3.5.

      1. Go to http://free.premierbiosoft.com. Click on Beacon Designer [Free Edition]. Then click on launch Beacon DesignerTM Free Edition.

      2. Click the SYBR Green option and enter the left primer sequence in the box for ‘Sense primer.’ Enter the right primer sequence in the box for ‘Anti-sense primer.’ Click ‘Analyze.’

      3. Beacon Designer free edition allows you to visualize secondary structures that can form between primers or primer pairs. An example of the secondary structure analysis is shown in Figure 6.

        Note: If self-dimers or cross dimers cannot be avoided, choose primers with the highest -ΔG (meaning the least negative number, the one closest to zero). Redesign primers with ΔGs more negative than -3.5 kcal/mol. If hairpins cannot be avoided, steer clear of hairpins that involve a 3’ end, and use UNAFold software to determine the melting temperature of the structure.



      Figure 6. Images of Beacon Designer webpage for analyzing the secondary structure of SYN1 primer.


    5. Use UNAFold software to check amplicon secondary structures.

      1. Once the primers have been checked for secondary structures, an additional QC step is needed to verify the amplicon’s secondary structures using UNAFold software by Integrated DNA Technologies (IDT).

      2. Go to: https://www.idtdna.com/UNAFold. Copy the amplicon (include both forward and reverse primers) into the sequence box. Change the annealing temperature to 60°C and the magnesium concentration to 3 mM. Click submit.

      3. Evaluate the structures that are displayed for the amplicon by checking for the ΔG values, which should not be -3.5.

    6. In-silico PCR (https://genome.ucsc.edu/cgi-bin/hgPcr): This is a QC step for verification of the exon spanning of the designed forward and reverse primers and also to cross verify the Tm and product length of the primer sets (Figure 7).

      1. Insert the forward and the reverse primer for each gene into the blank spaces.

      2. It is also important to set the target to GENCODE, which shows cDNA sequence, whereas genome assembly shows genomic DNA and allows to verify of intron spanning primer sequences. https://genome.ucsc.edu/FAQ/FAQgenes.html#ens.

      3. Select submit, to study results of in-silico PCR.



      Figure 7. In-Silico PCR analysis and result for SYN1 in UCSC browser.


    7. All the primers are designed and ordered at a 40 nmol scale. Primer stocks are re-constituted at 100 µM in nuclease-free water and dilutions at 10 µM are the working concentration.


  6. Test primer efficiency

    It is important to test primer efficiency for each primer pair by creating a standard curve with five serial dilutions of your cDNA template, e.g., 1:5 dilution. The primer efficiency should lie between 90-110%. The standard curve should cover the Ct value of the experimental value (Figure 8).

    1. The primer (forward and reverse) concentrations are 300 nM and the cDNA concentration is set for a 5-fold serial dilution (12.5 ng, 2.5 ng, 0.5 ng, 0.1 ng, 0.02 ng) for a 5 µL reaction volume as triplicates in a 384-well plate.

    2. The primer efficiency is calculated using the Excel template available at https://toptipbio.com/calculate-primer-efficiencies/.



    Figure 8. Primer efficiency calculation of genes involved in different timepoints of Ngn2 cortical neuron differentiation.

    OCT4 is tested for its efficiency with iPS cells.


  7. Plating of primers for multi-well SYBR Green qPCR array for cortical neuron differentiation

    For this array, we designed SYBR Green primers for 27 genes with two housekeeping genes. We wanted to capture pluripotency genes, neuro-precursor genes, and genes that are upregulated in cortical neurons. We also looked for synaptic markers and astrocyte gene expression. We used triplicates for each gene in a 96-well optical plate to study the expression (ΔΔCt) for each gene along with negative control (NC) and reverse transcriptase control (RTC). The working concentration of the primers diluted in nuclease-free water was 10 μM.

    1. Prepare the arrays for the PCR run at least a day in advance for the primers to dry out overnight.

    2. Plate primers according to the design of the 96-well, in triplicates (Figure 9). Pre-mix the forward and reverse primer for each gene and add to the corresponding well. The calculation for the amount of primer to be added to each well is shown in Step 3.



      Figure 9. 96-well plate template design for the multi-well qPCR reaction.


    3. Primer volume:

      1. Primer concentration in 20 μL qPCR reaction volume.

      2. Total reaction volume of qPCR reaction per well of 96 well plates: 20 µL.

      3. Primer volume (from 10 µM working solution): 0.6 µL (forward primer) + 0.6 µL (reverse primer) to reach 300 nM per reaction.

      4. Total: 1.2 µL of pre-mixed primers per gene per well.

      5. The primer mix is prepared in triplicates per gene. The amount of primer mix is (0.6 μL + 0.6 μL) × 4 times (includes 1 extra reaction for pipetting error). Prepare mix depending on the number of plates that are prepared.

      Optional: For plating of 96- or 384-well plates, pipetting can be facilitated by using PlateR (Biosistemika), a tablet-based visual support to pipette samples into wells.

    4. CRITICAL. Dispense the primer mix at the bottom of the well and spin down the plate for the primer mix to settle at the bottom of the well.

    5. Let the primer-coated plates dry overnight at room temperature in a Tupperware container to reduce the contamination.

    6. Time consideration: Coating of primers for a single 96-well plate takes about 20–30 min.

      Note: Do the primer coating for the array design after optimization in large batches. Plates can be stored at room temperature for several months, e.g., in sealed plastic bags.

    7. The concentration of primers and cDNA was determined as 300 nM and 2 ng (calculated for 96-well) based on the primer efficiency experiments (done in a 384-well plate) for each gene primer pair.


  8. qPCR Analysis (PowerUpTM SYBRTM Green Master Mix [Thermo Fisher, catalog number: A25780])

    PCR reaction mix:

    1. Since the primers are already pre-coated onto the plates, only the SYBR Green master mix, cDNA template, and nuclease-free water are combined as a master mix and added to each well. Since the pre-coated primers dry out, the master mix is made only with reagents shown in Table 4, which would be total volume of 20 μL.

    2. Before adding to the plate, the reaction mix is mixed thoroughly and spun down to avoid air bubbles. The plate should be kept on ice while adding the reaction mix. Calculate 1–2 extra reactions to account for pipetting errors. For a 96-well plate reaction, e.g., prepare a 98× reaction mix (Table 4).


      Table 4. Reaction mix for the multi-well Qpcr.

      Components Volume (each well)
      SYBR Green master mix 10 µl
      cDNA template 2 ng
      Nuclease free water variable
      Total reaction mix 20 µl


    3. Once the reaction mix is added, tightly seal the 96-well plate with the MicroAmp Optical adhesive film and centrifuge them briefly to remove any air bubbles and to bring all of the reaction mix to the bottom of the well.


  9. Plate set-up in QuantStudio 6 Flex and link to the software

    1. Once the plate is ready for the qPCR run, open the QuantStudio software v1.7.1 (link mentioned in the software section).

    2. PCR reaction set-up (for Tm of Primers more than 60°C):

      1. Spin down the plate after adding the reaction mix and then place it in the QuantStudio 6 Flex.

      2. The thermal cycling settings are set according to the Table 5 below for the Tm of primers between 63–66°C. Annealing temperature should be approximately 3°C lower than Tm.


        Table 5. Thermal cycling settings for the PCR reaction.

        Step Temperature Duration Cycles
        UDG activation 50°C 2 min Hold
        Dual-LockTM Taq DNA polymerase 95°C 2 min Hold
        Denature 95°C 15 s 40
        Anneal/ extend 60°C 1 min


      3. The instrument should be set for the default dissociation step as shown in Table 6.


        Table 6. Setting for the dissociation step.

        Step Ramp rate Temperature Time
        Denature 1.6°C/s 95°C 15 s
        Anneal 1.6°C/s 60°C 1 min
        Dissociation 0.15°C/s 95°C 15 s


      4. Once the run is complete the Ct data can be analyzed by clicking on analyze and the data can be exported as an excel file by clicking on the excel sheet.

      5. The plate setup in Quantstudio 6 is illustrated in Figure 10. For detailed plate set up, run parameters, and analysis, follow the User Guide for the QuantStudio software attached: https://tools.thermofisher.com/content/sfs/manuals/4489822.pdf.



      6. Figure 10. Workflow representation of setting up the plate in a QuantStudio 6 Flex software.

Data analysis

For the data analysis, we used the QuantStudio software to analyze the Ct values of each gene and the variability of replicates. The acceptable cycle difference should not be more than 0.5 cycles. The housekeeping genes B-ACTIN or GAPDH are used as the loading controls for ΔΔCt calculation (Livak and Schmittgen, 2001) of genes comparing the Day 0, Day 15, and Day 30 cortical neurons Ct values to the iPSCs Ct value. The negative template control should not show amplification (The link for the calculation of ΔΔCt in Excel is https://1drv.ms/x/s!AgUabBW4Y2yQgZEon-ZKuwMJ4PhaLQ).


As shown in Table 7, to calculate the average of 2-ΔΔCt fold change of genes, we initially compared the Ct of the gene of interest with the house-keeping gene (control), B-ACTIN, and the difference between them is the ΔCt. Next, to calculate the ΔΔCt, take an average of the ΔCt values of the iPSCs. When we subtract the ΔCt values of each sample from the average (ΔCt)iPSCs, we get the ΔΔCt. With the ΔΔCt values, we calculate the 2-ΔΔCt and the average of 2-ΔΔCt. This shows the fold change in the expression of genes in each sample relative to their expression in iPSCs. To visually illustrate the changes in gene expression, we plotted a heatmap for samples at different time points using GraphPad Prism. The pluripotency markers OCT4 and NANOG are highly expressed in the iPSCs (Figure 10) and expression is drastically reduced and, in some instances, undetectable with the assay at Day 15 and 30 of the cortical neuronal differentiation. Markers for early mature cortical, neuronal, and synaptic proteins show an increase in their expression on day 15 and 30 samples compared to the iPSCs (Figure 11). Both housekeeping genes B-ACTIN and GAPDH showed comparable results.


Table 7. ΔΔCt calculation of NANOG gene.



Figure 11. Fold change of gene expression using β-Actin as housekeeping gene comparing iPSCs with Day 0, Day 15, and Day 30 cortical neurons for pluripotency markers (OCT4 and NANOG), early neuronal markers (SOX2 and NESTIN), mature cortical neuronal markers (POU3F2 and TBR1) and synaptic markers (BASSOON and SYNAPSIN).


Summary:

In summary, we describe a comprehensive and economical protocol for a versatile multi-well SYBR Green qPCR protocol. Single-tube SYBR Green qPCR is a standard procedure in many labs to assess gene expression and there are also commercial platforms available for SYBR Green arrays. However, our protocol focusses on an efficient way to analyze multiple genes by building a multi-well qPCR array that can be customized and stored for several months at room temperature. These arrays are ideally suited to monitor iPSC differentiation protocols into various cell types and these arrays can be easily customized.

Acknowledgments

The study was supported by the California Institute for Regenerative Medicine (CIRM) Bridges program (TB1-01195).

Competing interests

Nothing to disclose.

Ethics

The work was approved under Stem Cell Research Oversight protocol SCRO-754 to use the WTC11 human-induced pluripotent stem cell for the Ngn2 differentiation.

References

  1. Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N. and Patel, H. R. (2005). Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 5(2): 209-219.
  2. Arikawa, E., Quellhorst, G., Ying, H., Pan, H. and Yang, J. (2011). RT2 ProfilerTM PCR arrays: Pathway-focused gene expression profiling with qRT-PCR. BioTechniques 43(5).
  3. Boone, D. R., Micci, M. A., Taglialatela, I. G., Hellmich, J. L., Weisz, H. A., Bi, M., Prough, D. S., DeWitt, D. S. and Hellmich, H. L. (2015). Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury. PLoS One 10(5): e0127287.
  4. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt Method. Methods 25(4): 402-408.
  5. SantaLucia, J., Jr. (1998). A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics.Proc Natl Acad Sci U S A 95(4): 1460-1465.
  6. Thornton, B. and Basu, C. (2011). Real-time PCR (qPCR) primer design using free online software.Biochem Mol Biol Educ 39(2): 145-154.
  7. Wang, C., Ward, M. E., Chen, R., Liu, K., Tracy, T. E., Chen, X., Xie, M., Sohn, P. D., Ludwig, C., Meyer-Franke, A., et al. (2017). Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening. Stem Cell Reports 9(4): 1221-1233.
  8. Zhang, Y., Pak, C., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., Xu, W., Yang, N., Danko, T., Chen, L., Wernig, M. and Sudhof, T. C. (2013). Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78(5): 785-798.

简介

[摘要] 为了优化基于干细胞的体外建模应用的分化方案,必须评估分化过程中基因表达的变化。这允许控制其分化为目标细胞类型的效率。虽然 RNA 转录组学提供了更大规模的细节,但在优化过程中包含此类分析的时间和成本令人望而却步。相比之下,单个基因的表达分析既繁琐又冗长。
在这里,我们开发了一个多功能且具有成本效益的 SYBR Green 阵列,该阵列包含 27 个标记以及两个管家基因,以快速筛选人类诱导多能干细胞 (iPSC) 分化为兴奋性皮层神经元的效率。我们首先通过文献检索确定了阵列的相关多能性、神经祖细胞和神经元标记,并设计了产物大小为 80-120 bp 长度、退火温度为 60°C 和最小预测二级结构的引物。我们在 96 孔板上发现了组合的正向和反向引物,并将它们干燥过夜。这些板可以提前分批制备,并在室温下储存直至使用。接下来,我们将 SYBR Green 预混液和互补 DNA (cDNA) 一式三份添加到板中,在Quantstudio 6 Flex 上运行定量 PCR (qPCR),并使用QuantStudio 软件分析结果。
我们比较了皮层分化过程中四个不同时间点在 96 孔格式中多能性、神经祖细胞、皮层神经元和突触标记的基因表达。我们发现在预分化的前三天内多能性基因急剧减少,并且随着时间的推移神经元标志物和突触标志物稳定增加。总之,我们构建了一个可定制、快速、中等通量且具有成本效益的基因表达阵列,非常适合优化基于干细胞的体外建模的分化方案。

[背景] 实时定量 PCR (qPCR) 技术检测靶核酸序列的扩增,被认为是灵敏的、可重复的和特异的(Arya et al ., 2005) 。
在这里,我们使用多孔 qPCR 分析通过 SYBR Green 技术扩增多个基因(Arikawa et al ., 2011) 。 SYBR Green 是一种 DNA 结合染料,可与双链 DNA (dsDNA) 非特异性结合(Boone et al ., 2015) 。
基于 RT 2 Profiler TM阵列(Arikawa et al ., 2011) ,我们设计了一个多孔 SYBR Green qPCR panel,通过强制表达 Neurogenin 2 来分析参与皮层神经元与人类 iPSCs 分化的基因的表达。 (Ngn2),它是一种神经元转录因子,支持人类胚胎干细胞或 iPSCs 的神经元分化为皮质样神经元(Zhang et al ., 2013) 。在这里,我们使用了人类 iPSC 细胞系,该细胞系将多西环素诱导型小鼠 Ngn2 转基因工程化到安全港基因座中(Wang et al ., 2017) 。我们收集了不同时间点的细胞(iPSC、第0 天前神经元、第15天和第30 天皮质神经元),并在单个 96 孔板上分析了标记多能干细胞、中间神经祖细胞和成熟皮质神经元的基因。扩增子/引物设计范围为 80-120 bp,Tm 介于 63°C 和 66°C 之间。多孔 SYBR Green qPCR 检测的内部制备允许根据需要定制引物以适应特定实验和检测修改。这种多孔 SYBR Green qPCR 检测可用于对任何一组感兴趣的基因进行定量分析。 用于通路分析的不同 RT2 分析器阵列可商购获得,但是,它们都没有经过优化以跟踪源自 iPSC 的皮质神经元的成熟。我们表明,多孔 SYBR Green qPCR 很容易适应实验室的定制。它是一个经济的平台,非常适合优化体外干细胞建模的分化方案。

关键字:人类 iPSC, 诱导多能干细胞, 皮质神经元, 神经元分化, SYBR Green, 定量 PCR, 多孔 qPCR, 引物设计

材料和试剂

A. RNA提取

1. 均质器旋转柱(Thermo Fisher Scientific Life Technologies目录号: 12183-026

2. 2-巯基乙醇(Sigma-AldrichAldrich Chemistry目录号: M2650

3. DNase I,扩增级(Thermo Fisher ScientificInvitrogen目录号: 18068015)

4. 乙醇,分子级(Thermo Fisher Scientific目录号: BP2818500

5. PureLinkR RNA mini 试剂盒(Thermo Fisher Scientific Life Technologies目录号: 12183025

6. RNase awayThermo Fisher Scientific Life Technologies目录号: 10328011 )

 

B. cDNA逆转录

1. MicroAmp 8管带(Thermo Fisher Scientific Applied Biosciences目录号: A30589

2. 高容量 cDNA 逆转录试剂盒(Thermo Fisher Scientific Invitrogen目录号: 4368814

 

C. 多孔PCR反应

1. MicroAmp Optical 96孔反应板(Thermo Fisher Scientific Applied Biosystems目录号: N8010560

2. MicroAmp 光学胶膜(Thermo Fisher Scientific Applied Biosystems,目录号:4311971

3. 无核酸酶水(US Biological LifeSciences目录号: W0900

4. PowerUp TM SYBR TM Green master mixThermo Fisher Scientific Applied Biosystems目录号: A25780

 

D. 消耗品

1. 1.5 mL 0.6 mL RNase 微量离心管

2.  RNase 过滤移液器吸头(P1000P200P20 P2

 

E. 其他

1. 10 × PBS,分子级(Fisher Scientific,目录号:J75889K2

2. AccutaseThermo Fisher Scientific,目录号:NC9464543

3. 装有湿冰的桶

4. 个人防护装备(手套、实验室外套、护目镜)

 

设备

 

1. Revco Ultima II 超低温-86°C冰箱(Thermo Scientific,目录号:ULT2586-9

2. 微量离心机5415CEppendorf目录号: M7282

3. 冷冻离心机(Beckman Coulter目录号: GS6 Allegra

4. 迷你离心机(Fisher Scientific目录号: 05-090-100

5. 水浴(Thermo Fisher ScientificCole Parmer,目录号:TSGP20

6. MiniAmp TM热循环仪(Applied BiosystemsThermo Fisher Scientific目录号: A37834

7. NanoDrop 分光光度计(Thermo Fisher Scientific目录号: 13-400-525

8. QuantStudio 6 FlexApplied BiosystemsThermo Fisher Scientific目录号: 4485691

9. 可选:PlateR 视觉移液辅助片剂(Biosistemika目录号: P-10

 

软件


1. 信标设计器(Premier Biosofthttp://www.premierbiosoft.com/qOligo/Oligo.jsp? PID=1

2. 计算机 PCR 预测(UCSC 基因组浏览器, https: //genome.ucsc.edu/cgi-bin/hgPcr

3. Primer3 软件(怀特黑德生物医学研究所、Steve RozenMaido RemmTriinu Koressaar Helen Skaletskyhttps: //bioinfo.ut.ee/primer3-0.4.0/

4. QuantStudio Flex 6-v1.7.1(应用生物系统,ThermoFisher Scientifichttps: //www.thermofisher.com/us/en/home/global/forms/life-science/quantstudio-6-7-flex-software.html

5. UNAFold(集成 DNA 技术, https: //www.idtdna.com/UNAFold )。创建一个帐户以使用该软件

 

程序

 

A. Ngn2 引导的皮层神经元样本采集

根据 Wang等人的方案,以 1.5 × 10 5 个细胞接种的 iPSC 12 孔板(每 cm 240,000 个细胞)中分化 (2017) 使用 Ngn2 诱导细胞系。媒体每隔一天更换一次。如图 1 所示,分别在第 -3天(多西环素诱导前 3 天) 、第 0 天、第 15 天和第 30 天收集 iPSC、前神经元、第 15 天和第 30 天皮质神经元的细胞,用于此 SYBR Green 多孔阵列的插图。 12 iPSC 板的汇合孔可产生 3 × 10 6 -4 × 10 6 细胞,RNA 产量约为 8-10 μg RNA。对于前神经元,2 × 10 6 -3 × 10 6 个细胞 RNA 产量范围为 5-6 μg ,对于神经元(15 天和第 30 天),RNA 产量范围为 1 - 3 μg,从 1 × 10 6 -2 × 10 6 细胞。

 

 

1 Ngn2 引导的皮层神经元分化和 RNA 提取样本收集的时间表。比例尺:100 μm

 

1. 为分离细胞进行样本采集,吸出旧培养基,直接在 12 孔板的每孔中加入 400 μL Accutase,并在 37°C 下孵育细胞 3-5 分钟以将其提起。

2.  400 μL 1 × PBS 添加到 Accutase 中,上下移取细胞溶液以分离,并将细胞悬液转移到 1.5 mL Eppendorf 管中。用额外的 400 μL 1 ×PBS清洗井,以收集剩余的细胞并添加到同一 Eppendorf 管中。

3. ×离心细胞悬液 g 4°C 下保持 5 分钟。

4. 吸出并丢弃上清液。

5.  1 mL 1 × PBS 上下吹打数次清洗细胞沉淀,并再次以 15,000 ×离心 g 4°C 下保持 5 分钟。

6. 吸出上清液,同时保持在湿冰上。

7. 继续进行 RNA 提取或将细胞沉淀储存在 -80°C

 

B. RNA 提取和纯化(PureLinkR RNA Mini Kit, Thermo Fisher, Life Technologies, 12183025

准备:

1.  70% 乙醇和 RNase Away 清洁工作台。

2. 使用无 RNase 屏障过滤器吸头进行 RNA 提取。

3. 首次使用 Wash Buffer II 之前:

a.  60 mL 96-100% 乙醇直接添加到洗涤缓冲液瓶中。

b. 选中洗涤缓冲液 II 标签上的框以表明已添加乙醇。

c. 在室温下用乙醇储存洗涤缓冲液 II

4. 为每次纯化制备含有 1% 2-巯基乙醇的新鲜裂解缓冲液。在化学罩下,每 300 μL 裂解缓冲液添加 3 μL 2-巯基乙醇。

 

程序

1.  -80°C 冰箱中取出细胞沉淀并将其置于冰上(在室温下执行后续步骤)。

2. 向细胞沉淀中加入 300 μL 裂解缓冲液。

3. 高速涡旋 10 秒,直到细胞沉淀完全混合。

4. 将裂解物转移到插入无 RNase Eppendorf 管中的匀浆器离心柱中,并以 12,000 ×离心 g 2 分钟。离心后取出并丢弃均质器盒。

5. 在细胞匀浆中加入 300 μL 70% 乙醇。

6. 涡旋以彻底混合并分散加入乙醇后可能形成的任何可见沉淀物。

7. 将多达 700 μL 的样品转移到旋转墨盒(带有收集管)中。

8. 在室温下以 12,000 × g离心15 秒。丢弃流出液并将旋转筒重新插入同一收集管中。

9.  700 μL 的洗涤缓冲液 I 添加到旋转盒中。 12,000 ×离心机 g在室温下保持 15 秒。丢弃流通和收集管。将离心筒放入新的收集管中。

10.  500 μL 的洗涤缓冲液 II(辅以乙醇)添加到旋转筒中。

11. 12,000 ×离心机 g在室温下保持 15 秒。丢弃流出液并将旋转筒重新插入同一收集管中。

12. 重复步骤 10-11 一次。

13. ×离心离心柱 g 2 分钟以干燥含有 RNA 的膜。

14. 丢弃收集管并将离心筒插入新的回收管中。

15.  30 μL 的无 RNase 水添加到旋转墨盒的中心。

16. 在室温下孵育 1 分钟。

17. ×条件下将离心柱离心 2 分钟 g在室温下将 RNA 从膜上洗脱到回收管中。

18. 将洗脱的 RNA 样本再次添加到同一个离心柱中,并重复步骤 16-17 以增加 RNA 样本的产量。

19. 使用 NanoDrop 分光光度计测定洗脱 RNA 的浓度。

20. 如图 2 中所述,RNA 浓度因细胞类型而异。

21. 关键一步! RNA 分装到不含 RNase 的试管中,每个试管含有 1 μg RNA,以避免冻融循环和 RNA 降解。 1 μg RNA 的等分体积直接用于 DNase I 处理。

注意:等分试样在储存前用 DNase I 处理。

22.  RNA 纯化后直接进行 DNase I 处理或在 -80 °C 下冷冻等分试样

23. 分装 RNA cDNA 样品以避免冻融循环和样品降解是绝对关键的。

 

 

2. 不同细胞类型通过皮层神经元分化的工作流程和计算,具有停止点和存储。 

 

C. DNase I Treatment Thermo Fisher ScientificInvitrogen目录号: 18068015

1. 将表 1 中所示的项目组合在无 RNase 管中:

 

1 用于 DNase I 处理洗脱 RNA 的反应混合物

 

2. 混合并在室温下孵育 15 分钟。

3. 通过添加 1 热灭活 DNase I μL 25mM EDTA 加入经 DNase I 处理的 RNA 样品中,并将其置于 65°C 的水浴中 3 分钟。

4. 一旦 RNA 样品被 DNase I 热灭活,将它们放在冰上。

5. 继续进行逆转录或将等分试样储存在 -80°C

 

D. cDNA逆转录High-Capacity cDNA Reverse Transcription KitThermo FisherInvitrogen目录号: 4368814

将提取的 RNA 转化为 cDNA 用于 qPCR 扩增。作为对照,准备一个逆转录对照,在反应混合物中不添加逆转录酶。

根据表 2 制备反应混合物(RT 混合物)。将来自上一步的1 μg RNA10 μL 体积)与反应混合物(表 2)混合,使 cDNA 的最终体积为 20 μL反应。

 

2 用于 cDNA 逆转录反应的反应混合物。

 

1. cDNA RT反应的制备:

a. 准备好 RT 混合物,轻轻混合,然后放在冰上。

b. 移液器 10 µ L RT 预混液到 MicroAmp 8 管条中。

c.  10 μL DNase I 处理的 RNA 样品 (1 μg ) 添加到反应混合物中,并通过上下吹打数次进行混合。

笔记:

i. 必须准备一个包含 RNA 输入但不包含 Multiscribe TM逆转录酶的试管。这用作逆转录酶对照 (RTC),它可以确认没有基因组 DNA 被扩增。

ii. 移液时不要引入气泡。

d. 将试管密封并离心以降低内含物并消除气泡。

e. 将试管置于冰上并将它们装入 MiniAmp TM热循环仪。

2. 逆转录热循环条件(表 3):

a. 反应体积设置为 20 µL

b. 将反应管装入热循环仪。

c. 热循环仪程序设置为 RUN

d. 循环完成后,在 20 μL cDNA 中加入 80 μL 无核酸酶水,工作浓度为 10 ng/μL,将 cDNA 储存在 -80°C

e. 关键一步!分装样品以避免冻融循环和 cDNA 降解。

 

3. cDNA 反应的热循环仪条件。

 

E. 引物设计

我们使用既定方案Thornton Basu2011)设计了 SYBR Green 引物。本部分引物参数为:产物大小:80-120 bp,产物熔解温度(Tm):63-66 ,二级结构ΔG :不大于-3.5GC百分比:35-80%,最优65%引物以跨内含子的方式设计,因此正向和反向引物不在同一个外显子中。这可以避免基因组 DNA 的污染。

 

1. 步骤1:从美国国家生物技术信息中心(NCBI)网站获取FASTA格式的目的基因序列: http ://www.ncbi.nlm.nih.gov  

a. 从搜索所有数据库的下拉菜单中选择核苷酸选项。

b. 在搜索框中输入感兴趣的基因名称或序列 ID,然后单击搜索。

c. 由于皮质神经元与人类 iPSC 不同,要么在搜索栏中提及智人以及基因名称,要么在单击搜索后从网页左上角的物种过滤器中选择智人。

d. 单击 RefSeq 转录本并选择 FASTA 选项,然后单击应用。

2. 步骤2:获得所选基因的FASTA序列后,使用Primer3软件设计引物: https ://bioinfo.ut.ee/primer3-0.4.0/ (圣卢西亚,1998 年),图 3

注意:默认值的解释在网页中给出: https ://bioinfo.ut.ee/primer3-0.4.0/input-help.htm

 

 

3  Primer3 网页截图显示了设计SYNAPSIN1 ( SYN1 ) 基因引物的参数和条件。

 

a. 将感兴趣的基因的 FASTA 格式序列复制并粘贴到 Primer3 引物设计页面上提供的框中。

b. 选择左侧引物:此选项留空,以便软件选择引物。

c. 挑选杂交探针:此选项留空。本实验不需要。

d. 选择正确的引物:如果此选项留空,Primer3 程序将选择正确的引物。

e. 序列 ID(基因名称):这是为了识别序列的引物。对应于序列设置专有名称。

f. 目标(感兴趣基因的序列区域):在查看 CDS 和外显子区域(如图 4 所示)后,针对目标输入序列中的特定核苷酸位置,然后是两侧的核苷酸数量围绕该特定区域设计的引物。图 4 显示了如何进入目标区域的示例。

 


4 指示外显子位置的 NCBI 网页图像

 

g. 引物 Tm:这是 50% 的引物与 DNA 模板杂交的温度。对于本实验,所有引物均设计在 63-66°C Tm 范围内。

h. 最大 Tm 差值:输入值为 2

i. 热力学参数表:Primer3 使用这些公式计算熔解温度。将方法设置为 SantaLucia (1998)

j. 产物 Tm:这是 50% 的扩增子是 ssDNA 时的温度。将最佳值设置为 50

k. Primer GC:这是允许的鸟嘌呤和胞嘧啶 (GC) 的最小和最大百分比。引物的GC含量用于确定引物的解链温度,可用于预测退火温度。将值设置为最小值:35,最佳值:65,最大值:80

l. 最大自互补:引物不应自互补或相互互补。自互补引物形成自二聚体或发夹结构。输入值为 4

m. 最大 3' 自互补:由于聚合酶在寡核苷酸的 3' 末端添加碱基,引物的 3' 末端不应相互互补,因为会产生引物二聚体。输入值为 3

n. Max #N:这是 Primer3 在设计引物时可以考虑的最大未知碱基数。将值设置为 0

o. Max Poly-X:引物中允许的最大单核苷酸重复数。长单核苷酸重复可以促进错误启动。输入值为 3

p. 内部目标罚分和外部目标罚分:如果引物需要设计为与某个区域重叠,则使用此选项。保留为默认值。

q. First Base Index:此参数告诉 Primer3 输入序列中的第一个碱基是哪种编程索引类型。保留为默认值。

r. GC Clamp:定义左右引物 3' Gs Cs 的具体数量。将该值保留为 0

s. 浓单价阳离子:这是 PCR KCl 盐的毫摩尔浓度。输入值为 50 µM

注意:根据 SantaLucia (1998),假设一价阳离子的浓度为 50 μM,二价阳离子的浓度为 3.5 mM。其他文献表明单价阳离子的范围为 20 100 μM,二价阳离子的范围为 1.5 5 mM

t. 盐校正公式:ΔG Tm 等因素会影响 PCR 性能并改变引物对的效率。 Primer3 首选 SantaLucia (1998) 盐配方。该公式旨在适应与序列无关但取决于寡核苷酸长度的盐校正。

u. 浓二价阳离子的浓度:这是 PCR 混合物中存在的二价盐的浓度。将值设置为 3.5 毫米。

v. dNTPs:通常建议使用 200 µM dNTP 浓度,以便 Taq 聚合酶在常规 PCR 中有效发挥作用。一些 SYBR Green 预混液带有 TaqKClMgCl 2 dNTP。这些在实验室中测试的混合物可提供最佳性能。输入值为 0.20 mM

w. 退火寡核苷酸浓度:用于计算寡核苷酸的熔解温度,这是 PCR 中退火寡核苷酸的纳摩尔浓度。保持默认。

x. 引物的目标函数惩罚权重:惩罚权重部分允许 Primer3 用户修改 Primer3 用于选择最佳引物组的标准。

引物的目标函数惩罚权重:

• Tm Lt = 1Gt = 1

尺寸 Lt = 1Gt =1

自互补 = 3

• 3' 自互补 = 3

• #N = 2

所有其他值 = 0

引物对的目标函数惩罚权重:

产品 TmLt = 1Gt = 1

• Tm 差异 = 2

任何互补 = 3

• 3' 互补 = 3

• Primer Penalty weight = 1

所有其他值 = 0

3. 分析引物:一旦所有的 Primer3 参数都设置好了,Primer3 就会设计引物对选项。

a. 输入所有选项后,按“Pick Primers”

b. 序列将显示在引物下方,并显示生成引物的详细信息,引物在序列内的位置由正向引物 >>>>>>> 和反向引物 <<<<<< 指示.使用 Beacon Designer TM免费版分析具有正确产物长度和 Tm 的第一个引物的二级结构。根据3 '值选择引物对,不大于3.003' 值是在引物对中形成的引物二聚体的测量值(图 5)。

 

 

5 Primer3 输出的图像,指示 3 ' 值,用于 SYN1 中的引物二聚体

 

4. 使用 Beacon Designer TM免费版检查引物二级结构。引物可接受的 ΔG 值不应超过 -3.5

a. 访问http://free.premierbiosoft.com 。点击 Beacon Designer [免费版]。然后点击启动 Beacon Designer TM免费版。

b. 单击 SYBR Green 选项并在感觉底漆框中输入左侧底漆序列。在反义引物框中输入正确的引物序列。点击分析

c. Beacon Designer 免费版允许您可视化可以在引物或引物对之间形成的二级结构。二级结构分析的示例如图 6 所示。

注意:如果无法避免自二聚体或交叉二聚体,请选择 -ΔG 最高的引物(即负数最小,最接近零的引物)。重新设计 ΔG -3.5 kcal/mol 更负的引物。如果无法避免发夹,请避开涉及 3' 端的发夹,并使用 UNAFold 软件确定结构的熔化温度。

 

 

6  用于分析 SYN1 引物二级结构的 Beacon Designer 网页图像。

 

5. 使用 UNAFold 软件检查扩增子二级结构。

a. 一旦检查了引物的二级结构,就需要额外的 QC 步骤来使用 Integrated DNA Technologies (IDT) UNAFold 软件验证扩增子的二级结构。

b. 访问: https ://www.idtdna.com/UNAFold 。将扩增子(包括正向和反向引物)复制到序列框中。将退火温度更改为 60°C,将镁浓度更改为 3 mM。点击提交。

c. 通过检查不应为 -3.5 ΔG 值来评估为扩增子显示的结构。

6. In-silico PCR ( https://genome.ucsc.edu/cgi-bin/hgPcr ):这是一个 QC 步骤,用于验证设计的正向和反向引物的外显子跨度,并交叉验证 Tm 和产物长度的引物组(图 7)。

a. 将每个基因的正向和反向引物插入空白处。

b. 将目标设置为显示 cDNA 序列的 GENCODE 也很重要,而基因组组装显示基因组 DNA 并允许验证内含子跨越引物序列。 https://genome.ucsc.edu/FAQ/FAQgenes.html#ens

c. 选择提交,以研究 in-silico PCR 的结果。

 

 

7 UCSC 浏览器中 SYN1 In-Silico PCR 分析和结果。

 

7. 所有引物均经过设计,并按 40 nmol 规模排序。引物原液在 100 µM 的无核酸酶水中重新配制10 µM 的稀释液是工作浓度。

 

F. 测试引物效率

测试每个引物对的引物效率很重要,方法是创建一个标准曲线,其中包含 5 cDNA 模板的连续稀释度,例如1:5 稀释度。引物效率应介于 90-110% 之间。标准曲线应覆盖实验值的 Ct 值(图 8)。

1. 引物(正向和反向)浓度为 300 nMcDNA 浓度设置为 5 倍连续稀释(12.5 ng2.5 ng0.5 ng0.1 ng0.02 ng),反应体积为5 µl ,一式三份一个 384 孔板。

2. https://toptipbio.com/calculate-primer-efficiencies/上提供的 Excel 模板计算引物效率 HYPERLINK "https://toptipbio.com/calculate-primer-efficiencies/"

 

 

8 Ngn2皮层神经元分化不同时间点相关基因的引物效率计算。 

使用 iPS 细胞测试 OCT4 的效率。

 

G. 用于皮层神经元分化的多孔 SYBR Green qPCR 阵列的引物电镀

对于这个阵列,我们为 27 个基因和两个看家基因设计了 SYBR Green 引物。我们想要捕获多能性基因、神经前体基因和皮质神经元中上调的基因。我们还寻找突触标记和星形胶质细胞基因表达。我们在 96 孔光学板中对每个基因使用一式三份来研究每个基因的表达 (ΔΔCt) 以及阴性对照 (NC) 和逆转录酶对照 (RTC)。在无核酸酶水中稀释的引物的工作浓度为 10 μM

1. 至少提前一天准备用于 PCR 运行的阵列,以便引物在一夜之间变干。

2. 根据 96 孔设计的板引物,一式三份( 9。预混合每个基因的正向和反向引物并添加到相应的孔中。步骤 3 中显示了要添加到每个孔中的引物量的计算。

 

 

9 用于多孔 qPCR 反应的 96 孔板模板设计。

 

3. 引物体积:

a. 20 μL qPCR 反应体积中的引物浓度。

b. 96 孔板每孔 qPCR 反应的总反应量:20 µL

c. 底漆体积(来自 10 µM 工作溶液):0.6 µL (正向底漆) +0.6 µL (反向底漆),每次反应达到 300 nM

d. 总计:每孔每个基因 1.2 µL 预混合引物

e. 每个基因一式三份制备引物混合物。引物混合物的量为 (0.6 μL + 0.6 μL) × 4 倍(包括 1 个额外的移液错误反应)。根据准备的盘子数量准备混合物。

可选:对于 96 384 孔板的电镀,可以使用 PlateR (Biosistemika) 促进移液,这是一种基于平板电脑的视觉支持,用于将样品移入孔中。

4. 关键。将底漆混合物分配在孔的底部并向下旋转板以使底漆混合物沉淀在孔的底部。

5. 让涂有底漆的板在室温下在特百惠容器中干燥过夜,以减少污染 

6. 时间考虑:单个 96 孔板的引物涂层大约需要 20-30 分钟。

注意:大批量优化后做阵列设计的底漆。板可以在室温下储存几个月,例如,在密封的塑料袋中。

7. 基于每个基因引物对的引物效率实验(在 384 孔板中进行),将引物和 cDNA 的浓度确定为 300 nM 2 ng(针对 96 孔计算)。

 

H. qPCR 分析(PowerUp TM SYBR TM Green Master Mix [Thermo Fisher目录号: A25780]

PCR反应混合物:

1. 由于引物已经预涂在板上,因此只有 SYBR Green 预混液、cDNA 模板和无核酸酶水作为预混液混合并添加到每个孔中。由于预涂底漆会变干,因此预混液仅使用表 4 中所示的试剂制成,总体积为 20 μL

2. 在加入板之前,将反应混合物彻底混合并向下旋转以避免气泡。加入反应混合物时,板应保持在冰上。计算 1-2 个额外反应以解决移液错误。对于 96 孔板反应,例如,准备 98 ×反应混合物(表 4)。

 

4 多孔 Qpcr 的反应混合物。

 

3. 加入反应混合物后,用 MicroAmp 光学胶膜紧紧密封 96 孔板并短暂离心以去除任何气泡并将所有反应混合物带到孔底。

 

I. QuantStudio 6 Flex 中的板设置和软件链接

1. 板准备好进行 qPCR 运行后,打开 QuantStudio 软件 v1.7.1(软件部分中提到的链接)。

2. PCR 反应设置(引物 Tm 超过 60°C):

a. 添加反应混合物后将板旋转下来,然后将其放入 QuantStudio 6 Flex

b. 对于 63-66°C 之间的引物的 Tm,根据下表 5 设置热循环设置。退火温度应比 Tm 低约 3°C

 

5  PCR 反应的热循环设置。

 

c. 仪器应设置为默认解离步骤,如表 6 所示。

 

6  解离步骤的设置。

 

d. 运行完成后,可以通过单击分析来分析 Ct 数据,并且可以通过单击 excel 表将数据导出为 excel 文件。

e. Quantstudio 6 中的印版设置如图 10 所示。有关详细的板设置、运行参数和分析,请遵循随附的 QuantStudio 软件用户指南: https ://tools.thermofisher.com/content/sfs/manuals/4489822.pdf

 

 

10 QuantStudio 6 Flex 软件中设置板的工作流程表示。

 

数据分析

 

对于数据分析,我们使用 QuantStudio 软件分析每个基因的 Ct 值和重复的变异性。可接受的循环差不应超过 0.5 个循环。管家基因 B-ACTIN GAPDH 被用作比较第 0 天、第 15 天和第 30 天皮层神经元 Ct 值与 iPSCs Ct 值的基因的 ΔΔCt 计算 (Livak and Schmittgen, 2001) 的加载对照。负模板控制不应显示放大(Excel ΔΔCt 计算的链接是https://1drv.ms/x/s!AgUabBW4Y2yQgZEon-ZKuwMJ4PhaLQ )。

如表 7 所示,为了计算基因 2 -ΔΔCt倍数变化的平均值,我们最初将感兴趣基因的 Ct 与看家基因(对照)、B-ACTIN 和差异 它们之间是ΔCt。接下来,要计算 ΔΔCt,取 iPSC ΔCt 值的平均值。当我们从平均 (ΔCt) iPSCs中减去每个样本的 ΔCt 值时,我们得到 ΔΔCt。使用 ΔΔCt 值,我们计算 2 -ΔΔCt 2 -ΔΔCt 平均值。这显示了每个样本中基因表达相对于它们在 iPSC 中的表达的倍数变化。为了直观地说明基因表达的变化,我们使用 GraphPad Prism 绘制了不同时间点的样本热图。多能性标记 OCT4 NANOG iPSCs 中高度表达(图 10),表达急剧下降,在某些情况下,在皮层神经元分化的第 15 天和第 30 天检测不到。与 iPSC 相比,早期成熟的皮质、神经元和突触蛋白的标志物在第 15 天和第 30 天样本中的表达增加(图 11)。管家基因 B-ACTIN GAPDH 都显示出相当的结果。

 

NANOG基因的 ΔΔCt 计算。

 

 

11 使用 β-肌动蛋白作为管家基因的基因表达倍数变化比较 iPSC 与第 0 天、第 15 天和第 30 天皮质神经元 多能性标志物(OCT4 NANOG)、早期神经元标志物(SOX2 NESTIN)、成熟皮质神经元标志物(POU3F2 TBR1)和突触标志物(BASSOON SYNAPSIN)。

 

摘要

总之,我们为多功能多孔 SYBR Green qPCR 协议描述了一个全面且经济的协议。单管 SYBR Green qPCR 是许多实验室评估基因表达的标准程序,也有可用于 SYBR Green 阵列的商业平台。然而,我们的协议侧重于通过构建可定制并在室温下存储数月的多孔 qPCR 阵列来分析多个基因的有效方法。这些阵列非常适合监测 iPSC 分化为各种细胞类型的方案,并且这些阵列可以轻松定制。

 

致谢

 

该研究得到了加州再生医学研究所 (CIRM) 桥梁项目 (TB1-01195) 的支持。

 

利益争夺

 

没有什么可透露的。

 

伦理

 

这项工作在干细胞研究监督协议 SCRO-754 下获得批准,使用 WTC11 人类诱导的多能干细胞进行 Ngn2 分化。

 

参考文献

 

1. Arya, M.Shergill, ISWilliamson, M.Gommersall, L.Arya, N. Patel, HR (2005)实时定量 PCR 的基本原理。 专家 Rev Mol Diagn 52):209-219

2. Arikawa, E.Quellhorst, G.Ying, H.Pan, H. Yang, J. (2011)RT2 Profiler TM PCR 阵列:使用 qRT-PCR 进行以通路为中心的基因表达谱分析。 生物技术435)。

3. Boone, DR, Micci, MA, Taglialatela, IG, Hellmich, JL, Weisz, HA, Bi, M., Prough, DS, DeWitt, DS Hellmich, HL (2015)创伤性脑损伤后激光捕获显微切割海马细胞的富集群体的通路聚焦 PCR 阵列分析。 公共科学图书馆一号10(5)e0127287

4. Livak, KJ 和施密特根, TD (2001)使用实时定量 PCR 2 -ΔΔCt方法分析相对基因表达数据。方法25(4)402-408

5. SantaLucia, J., Jr. (1998)聚合物、哑铃和寡核苷酸 DNA 最近邻热力学的统一视图。 Proc Natl Acad Sci USA 954):1460-1465

6. Thornton, B. Basu, C. (2011)使用免费在线软件进行实时 PCR (qPCR) 引物设计。 生化分子生物学教育392):145-154

7. Wang, C., Ward, ME, Chen, R., Liu, K., Tracy, TE, Chen, X., Xie, M., Sohn, PD, Ludwig, C., Meyer-Franke, A., et_ 2017)。 iPSC 衍生的人类神经元的可扩展生产,以通过高内涵筛选识别 Tau 降低化合物。干细胞报告9(4): 1221-1233

Zhang, Y., Pak, C., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., Xu, W.Yang, N.Danko, T.Chen, L.Wernig, M. Sudhof, TC (2013)从人类多能干细胞中快速单步诱导功能性神经元。 神经元785):785-798

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引用:Srinivasaraghavan, V. N., Zafar, F. and Schuele, B. (2022). Gene Expression Analysis in Stem Cell-derived Cortical Neuronal Cultures Using Multi-well SYBR Green Quantitative PCR Arrays . Bio-protocol 12(14): e4476. DOI: 10.21769/BioProtoc.4476.
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