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Gene Expression Analysis in Stem Cell-derived Cortical Neuronal Cultures Using Multi-well SYBR Green Quantitative PCR Arrays

VS Vasavi Nallur Srinivasaraghavan
FZ Faria Zafar
BS Birgitt Schüle

Published: Jan 5, 2022 DOI: 10.21769/BioProtoc.4283 Views: 1182

Edited by: Salma Merchant Reviewed by: Valerie Uytterhoeven

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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 search Induced pluripotent stem cells search Cortical neurons search Neuronal differentiation search SYBR Green search Quantitative PCR search Multi-well qPCR search Primer design search

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 economical 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

Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.

Category

Stem Cell > Pluripotent stem cell > Cell differentiation

Neuroscience > Nervous system disorders > Cellular mechanisms

Molecular Biology > DNA > Gene expression

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