Harnessing Applied Biosystems™ TaqMan™ Real-Time PCR Assays for research in profiling miRNA and gene expression in cystic fibrosis

 

Cystic fibrosis (CF) is a severe inherited disease characterized by multi-system manifestations and reduced life expectancy due to progressive lung disease and other complications. Classified as a rare disease, it affects approximately 100,000 people globally (1).

The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Over 2,000 mutations have been identified in CFTR, which may impair mRNA/protein expression, function, or stability (1). The CFTR protein functions as a cAMP-dependent, phosphorylation-activated anion channel that transports chloride and bicarbonate across the apical plasma membrane of epithelial cells. It also modulates the activity of other ion channels, such as the epithelial sodium channel (ENaC). The proper function of CFTR is crucial for maintaining the balance of ions and fluid in cells of various organs, including the sweat glands, airways, intestine, and pancreas. Its absence or dysfunction leads to impaired transepithelial ion and fluid balance, contributing to the symptoms of cystic fibrosis (1,2).

Novel therapies called CFTR modulators, capable of enhancing or restoring the function of the affected protein, have been developed recently, transforming the treatment landscape for cystic fibrosis. These include potentiators that enhance the open probability of CFTR channels, allowing for anion conductance, correctors that assist in the proper folding and trafficking of mutant CFTR proteins to the plasma membrane, stabilizers that increase the stability of mutant CFTR proteins at the plasma membrane, read-through agents and non-sense mediated decay (NMD) inhibitors that rescue CFTR mRNA and thereby, protein synthesis, and amplifiers that increase the amount of CFTR mRNA, leading to an increase in CFTR protein production (2). Due to the divergence in disease-causing mutations, some rare and ultra-rare mutations still lack efficient disease-modifying therapies. More research is needed to bridge this gap.

TaqMan^™ research assays are trusted for quantitative PCR (qPCR) due to their high specificity, reproducibility and sensitivity. In this article, we focus on the use of TaqMan^™ qPCR technologies for research in profiling gene and miRNA expression in cystic fibrosis.

CFTR gene expression analysis using TaqMan^™ qPCR probes

Treatment options are limited for CF patients with premature termination codons (PTCs) in the CFTR gene. eRF3a degraders, a class of small-molecule compounds that target eukaryotic release factor 3a (eRF3a), are investigated for their potential to rescue CFTR nonsense mutations by promoting PTC readthrough. Using cell lines that recapitulate this phenotype, a study investigated eRF3a degraders as a potential treatment for CF caused by PTCs in the CFTR gene. This approach demonstrates a promising therapeutic strategy for CF patients with PTC variants, offering a potential pathway to develop effective future treatments (3).

In this study, the researchers used TaqMan^™ qPCR assays for CFTR to assess CFTR mRNA levels. The results indicated the suppression of NMD and increased CFTR expression after treatment with eRF3a degraders (3).

miRNA expression profiling in cell line models of cystic fibrosis

MicroRNAs (miRNAs) are known to regulate CFTR gene expression and are, thereby, hypothesized to influence the various clinical manifestations of cystic fibrosis. A study investigating miRNA changes in two different CFTR mutant cell lines revealed that CFTR loss of function is linked to alterations in the miRNA network, which regulates genes involved in major CF comorbidities. A global miRNA profiling approach using TaqMan^® Array MicroRNA Cards identified changes in 41 differentially expressed miRNAs in CFTR mutant cell lines compared to a control cell line (4).

Profiling global miRNA expression can be a useful first step in identifying disease-specific miRNAs. However, miRNAs can be expressed over a wide range, warranting research tools that offer high sensitivity over a broad dynamic range. Using the Applied Biosystems^™ TaqMan^™ OpenArray^™ MicroRNA workflow, researchers can generate as many as 48 miRNA expression profiles from human, mouse, or rat samples in a single working day, starting with as little as 100 ng of input total RNA. This is unlike microarrays, which require several days and hundreds of nanograms of input RNA (5).

The TaqMan^™  OpenArray^™ MicroRNA Panel research workflow: The medium throughout TaqMan^TM Array MicroRNA Cards or the high-throughput TaqMan^™ OpenArray^™ MicroRNA Panels can be used for reverse transcription of up to 377 and 754 miRNAs, respectively.  Applied Biosystems^™ Megaplex^™ RT primers and real-time PCR instruments are used in this process. A preamplification step using Megaplex^™ PreAmp Primers increases miRNA concentration in the sample. For quantitation, TaqMan^™  Universal PCR Master Mix II is added to each sample. The mixtures are loaded into TaqMan^™  OpenArray^™ MicroRNA Panels or TaqMan Array MicroRNA Cards for the final real-time PCR step. The data are then analyzed using the appropriate software.

Click here to learn more about Applied Biosystems^™ solutions for research into miRNA profiling and gene expression analysis:

References:

1. Allen L, Allen L, Carr SB, Davies G, Downey D, Egan M, Forton JT, Gray R, Haworth C, Horsley A, Smyth AR, Southern KW, Davies JC. Future therapies for cystic fibrosis. Nat Commun. 2023 Feb 8;14(1):693. doi: 10.1038/s41467-023-36244-2. PMID: 36755044; PMCID: PMC9907205.
2. Lopes-Pacheco M. CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine. Front Pharmacol. 2020 Feb 21;10:1662. doi: 10.3389/fphar.2019.01662. PMID: 32153386; PMCID: PMC7046560.
3. Lee RE, Lewis CA, He L, Bulik-Sullivan EC, Gallant SC, Mascenik TM, Dang H, Cholon DM, Gentzsch M, Morton LC, Minges JT, Theile JW, Castle NA, Knowles MR, Kimple AJ, Randell SH. Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough. J Clin Invest. 2022 Sep 15;132(18):e154571. doi: 10.1172/JCI154571. PMID: 35900863; PMCID: PMC9479597.
4. Catellani C, Cirillo F, Graziano S, Montanini L, Marmiroli N, Gullì M, Street ME. MicroRNA global profiling in cystic fibrosis cell lines reveals dysregulated pathways related with inflammation, cancer, growth, glucose and lipid metabolism, and fertility: an exploratory study. Acta Biomed. 2022 Jul 1;93(3):e2022133. doi: 10.23750/abm.v93i3.12842. PMID: 35775757; PMCID: PMC9335447.
5. Applied BioSystems Brochure: MicroRNA: having a big impact on biology. PG1503-PJ9854-CO020249-TS-rebrand-TaqMan-comprehensive-microRNA-Broch-Corp-FLR (thermofisher.com)

For Research Use Only. Not for use in diagnostic procedures.