Absolute Quantification of mRNA Isoforms in Adult Stem Cells Using Microfluidic Digital PCR

Background

Muscle stem cells (MuSCs) reside beneath the basal lamina of skeletal muscle fibers and maintain a state of quiescence (de Morree and Rando, 2023). Upon injury, the MuSCs switch from quiescence to activation, giving rise to proliferating progenitors that will eventually differentiate and fuse with myofibers (Yin et al., 2013;Relaix et al., 2021). To understand the exit from quiescence and how the myogenic fate is determined, it is important to understand the factors that maintain or break the quiescent state The transcription factor Pax3 plays a key role in myogenesis. In the absence of Pax3, embryonic MuSCs fail to expand and migrate, and no muscle is formed, resulting in embryonic lethality (Relaix et al., 2005). Recent work revealed that thePax3gene generates multiple mRNA isoforms with different 3′UTRs in MuSCs from adult mice (Boutet et al., 2012; de Morree et al., 2019). One of the isoforms of Pax3 mRNA has a long 3′UTR containing microRNA binding sites, is translationally repressed by the microRNA 206, and is linked to quiescence (Figure 1). The other has a short 3′UTR lacking the microRNA binding sites, is not translationally repressed, and is linked to activation and cell cycle entry of MuSCs (de Morree et al., 2019). Therefore, it is important to understand Pax3 isoform expression patterns in MuSCs, as quantifying the two isoforms of Pax3 will contribute to understanding its role in MuSCs quiescence and activation.

Figure 1. Schematic figure depicting how the TaqMan probes, Pax3total and Pax3long, target their respective target sites on the Pax3 3′ terminal exon. Top: a schematic of thePax3gene with boxes denoting exons. The 3′ terminal exon is enhanced, and the open reading frame (ORF) coding part is highlighted, as well as the three polyadenylation sites (PASs) present in the 3′ untranslated region (3′UTR). Bottom: three major Pax3 mRNA isoforms are represented. All contain the complete ORF (not depicted) but differ in the length of the 3′UTR due to the selection of different PASs. These isoforms can be detected with TaqMan probes. The Pax3total TaqMan probes hybridize to sequences that are present in both long and short isoforms of Pax3. The Pax3long TaqMan probes hybridize to sequences that are present only in long isoform of Pax3. Each TaqMan probe contains a reporter dye and a quencher. During the polymerase reaction, the dye and the quencher are separated from the target sequence; as a result, fluorescence increases.

Quantitative real-time polymerase chain reaction (qRT-PCR) is often considered as the gold standard for measuring mRNA expression levels, despite many factors of variability such as RNA templates, inconsistent data analysis, and data normalization (Nolan et al., 2006). This method uses intercalating fluorescent dyes to assess transcript abundance during a PCR amplification. Though effective, it cannot measure exact copy numbers and requires a reference gene like GAPDH or HPRT to calculate relative expression levels. These reference genes are used because they are considered stably expressed in all cells. However, they may not be (Kozera and Rapacz, 2013). MuSCs undergo a 6-fold increase in size during activation (Rodgers et al., 2014;Brett et al., 2020). Thus, the change in cell volume may affect the interpretation of relative quantification by qRT-PCR, complicating the interpretation of how much Pax3 is expressed in cells. Instead, several adaptations have been made that enable single molecule quantification using a technique called digital PCR (Mao et al., 2019). In digital PCR, the total reaction volume is partitioned into small reaction chambers at a dilution so that 5%–65% of the small reaction chambers contain a single molecule of target cDNA. Then, a PCR amplification is performed to amplify those molecules, enabling the researcher to simply score for each small reaction chamber whether a PCR reaction took place or not. This digital assessment can then be calculated into an exact number of molecules present in the original reaction volume using Poisson statistics. Recent technologies that enable the formation of small droplets (droplet digital PCR) made digital PCR more routinely accessible (Hindson et al., 2013;Mao et al., 2019).

However, while droplet digital PCR enables the low-cost quantification of absolute mRNA levels, there is a variability between droplets that lowers the precision (Emslie et al., 2019), which is key when measuring low-abundant transcripts. Here, we use a parallel technology, microfluidic digital PCR (mdPCR), in which the sample is partitioned in microfluidic nanochambers (see Graphical overview). These nanochambers are uniform and enable highly reproducible amplification reactions, to enable the absolute quantification of low abundant transcripts with high accuracy. We previously applied this protocol to find that MuSCs isolated from lower hindlimb muscle expressed on average three molecules of Pax3 mRNA, two thirds of which were long isoforms. In contrast, MuSCs isolated from the diaphragm muscle expressed on average 10 molecules of Pax3 mRNA, one third of which were long isoforms (de Morree et al., 2019).

This protocol can also be used to quantify mRNA isoforms in other stem cell types, such as muscle-resident fibroadipogenic progenitors, which play important roles in homeostasis and repair of multiple tissues (Joe et al., 2010;Uezumi et al., 2010). The levels of mRNA isoforms can be quantified accurately across different cell states. Expression of disease genes along the progression of a disease can also be studied using mdPCR. Finally, the efficacy of therapies that change relative isoform abundance, such as exon-skipping for Duchenne Muscular Dystrophy, can be assessed with mdPCR (Verheul et al., 2016). Hence, this protocol can be used both in basic biology and translational studies.