Background

Quantitative reverse transcriptase PCR (qRT-PCR) is a fundamental laboratory technique to quantify the RNA gene expression of a biological sample. Previously, we developed a protocol that quantifies PCR products using an SYBR Green array in a 96-well format. SYBR Green is a dye that fluoresces when intercalated into double-stranded DNA (Srinivasaraghavan et al., 2022). As a region of interest is PCR-amplified, the amount of fluorescence is directly proportional to the amount of DNA content in the sample. This assay provides an accurate, cost-effective, and fast way to quantify the mRNA gene expression of a cell or tissue sample. Here, we optimized the throughput with a 384-well format, increasing the number of markers, simplifying and standardizing primer design, and automating pipetting of the plate to reduce manual pipetting errors and ensure volume accuracy. Each well uses 5 μL of reaction mix instead of the 20 μL in a 96-well plate, but the whole plate contains four times the number of wells. Thus, for the same volume of reagents used, one plate can output four times the data.

Scaling up the assay from 96- to 384-well plates requires optimization

However, qRT-PCR methods such as TaqMan technology require fluorescent reporters to characterize target genes and are challenging to scale up as they need different probes for each reaction. Bulk or single-cell RNA transcriptomics is time-consuming and expensive (Table 1). Processing RNA sequencing data can take several weeks, which is not optimal for monitoring gene expression, e.g., during a differentiation paradigm of induced pluripotent stem cells (iPSCs) into a specialized cell type. We improved primer design by using the command line version of Primer3 software. This avoids using the web interface, where user input errors can occur. Instead, the program uses 1) a text file containing all settings and primer conditions; 2) an input file containing the gene name, full gene sequence, and target region to amplify; and 3) an output file/folder containing compatible primers. Coating 384-well plates with primers is labor-intensive and yields potential user error. To mitigate this, we use one of three methods for plating primers: 1) use of a PlatR pipetting aid, which uses a tablet that lights up wells from underneath, 2) an E1-ClipTip adjustable tip-spacing electronic pipette, or 3) Integra ASSIST PLUS pipetting robot to pipette larger arrays with more plates. All three methods increase the throughput of generating coated plates and the consistency of volumes across all assays.

With the optimized format we describe in this protocol, we can assess 31 markers in triplicates on four samples on a single 384-well plate. Alternatively, 40 markers can be plated in triplicates on a plate with three samples. The number of wells needed for each will vary depending on the number of genes, replicates, and samples.

Human iPSC modeling

Patient-specific iPSC lines can be generated and differentiated to study cell processes, developmental defects, or disease mechanisms on a human genomic background. However, since differentiating these cells requires careful design of differentiation protocols, a method is needed to validate and confirm that this differentiation resembles that of in vivo tissue.

The use of many markers allows SYBR Green to be used to characterize many cell types. In contrast, the customizable plate allows flexible comparison of samples at different differentiation time points or between different cell types. Because most of the workflow for this SYBR Green protocol can be done within 1–2 days, the low turnaround time is ideal for characterizing cells at various time points during the differentiation process.

Optimization and validation of differentiation protocols using cell type–specific markers

The data analysis for RNA-seq methods can take some time and requires advanced bioinformatics tools. Our SYBR Green qRT-PCR gene expression can be completed in ~2 days, in which cells are harvested, cDNA synthesis is performed, and cDNA samples are quantified (Figure 1).

To show that the optimized primer design process can generate more markers and establish markers for optimizing current and future differentiation protocols, we have developed a library of markers to characterize multiple neuronal types at different developmental time points (Supplemental Table 1). This primer inventory includes cortical, striatal, and dopaminergic neurons and progenitors, pluripotency genes, astrocytes, hematopoietic precursors, microglia, retinal genes, synaptic markers, and germ layer markers.

Please note that we have saved Supplemental Table 1 and Supplemental Table 2 as individual files.

Table 1. Comparison between different RNA analysis tools