Selective measurement of α smooth muscle actin: why β-actin can not be used as a housekeeping gene when tissue fibrosis occurs

Veres-Székely, Apor; Pap, Domonkos; Sziksz, Erna; Jávorszky, Eszter; Rokonay, Réka; Lippai, Rita; Tory, Kálmán; Fekete, Andrea; Tulassay, Tivadar; Szabó, Attila; Vannay, Ádám

doi:10.1186/s12867-017-0089-9

Cited by 35 publications

(14 citation statements)

References 26 publications

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“…The results were evaluated using the standard curve method and were expressed as the fold of the control values, normalized to β-actin. Specifically designed primer pairs and qPCR conditions were applied to selectively determine the expression of mouse β-actin, TGF-β1, collagen 1α2, fibronectin, elastin, and tenascin C, as previously described [54,55].…”

Section: Western Blot Analysismentioning

confidence: 99%

Age-Dependent Chronic Lung Injury and Pulmonary Fibrosis following Single Exposure to Hydrochloric Acid

Biancatelli

Solopov

Dimitropoulou

et al. 2021

IJMS

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Exposure to hydrochloric acid (HCl) represents a threat to public health. Children may inhale higher doses and develop greater injury because of their smaller airways and faster respiratory rate. We have developed a mouse model of pediatric exposure to HCl by intratracheally instilling p24 mice (mice 24 days old; 8–10 g) with 2 µL/g 0.1 N HCl, and compared the profile of lung injury to that in HCl-instilled adults (10 weeks old; 25–30 g) and their age-matched saline controls. After 30 days, alveolar inflammation was observed with increased proteinosis and mononuclear cells in the bronchoalveolar lavage fluid (BALF) in both HCl-instilled groups. Young p24 animals—but not adults—exhibited higher NLR family pyrin domain containing 3 (NLRP3) inflammasome levels. Increased amounts of Transforming Growth Factor-β (TGF-β) mRNA and its intracellular canonical and non-canonical pathways (p-Smad2 and p-ERK) were found in the lungs of both young and adult HCl-instilled mice. Constitutive age-related differences were observed in the levels of heat shock protein family (HSP70 and HSP90). HCl equally provoked the deposition of collagen and fibronectin; however, significant age-dependent differences were observed in the increase in elastin and tenascin C mRNA. HCl induced pulmonary fibrosis with an increased Ashcroft score, which was higher in adults, and a reduction in alveolar Mean Alveolar Linear Intercept (MALI). Young mice developed increased Newtonian resistance (Rn) and lower PV loops, while adults showed a higher respiratory system resistance and elastance. This data indicate that young p24 mice can suffer long-term complications from a single exposure to HCl, and can develop chronic lung injury characterized by a stronger persistent inflammation and lesser fibrotic pattern, mostly in the airways, differently from adults. Further data are required to characterize HCl time- and dose-dependent injury in young animals and to identify new key-molecular targets.

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Section: Western Blot Analysismentioning

confidence: 99%

Age-Dependent Chronic Lung Injury and Pulmonary Fibrosis following Single Exposure to Hydrochloric Acid

Biancatelli

Solopov

Dimitropoulou

et al. 2021

IJMS

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“…The mRNA expression of the investigated genes was determined by real-time PCR using LightCycler 480 SYBR Green I Master enzyme mix on a Light Cycler 480 system (Roche Diagnostics, Mannheim, Germany). The nucleotide sequences and annealing temperatures of the primer pairs and the resulted PCR product lengths are shown in Additional file 2 [18]. Relative mRNA expression was determined by comparison with RPLP0 as internal control using the ∆∆Ct method [19].…”

Section: Rna Isolation Reverse Transcription and Real-time Rt-pcrmentioning

confidence: 99%

Role of IL-24 in the mucosal remodeling of children with coeliac disease

et al. 2020

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Background: Recently, involvement of IL-19, IL-20 and IL-24 has been reported in inflammatory diseases associated with tissue remodeling. However, their impact on the pathomechanism of coeliac disease (CD) is still completely unknown. Methods: Expression of IL19, IL20 and IL24 was measured by real-time RT-PCR, protein amount of IL-24, α smooth muscle actin (α-SMA) and fibronectin (FN) was determined by Western-blot analysis in the duodenal biopsies of therapy naive children with CD and controls. Localization of IL-24 and IL-20RB was investigated by immunofluorescent staining in the duodenal mucosa. Effect of recombinant IL-1β, TNF-α, TGF-β and IL-17 treatment on the expression of IL19, IL20, IL24 and their receptors was investigated by real-time RT-PCR in small intestinal epithelial cells (FHs74Int), in primary duodenal myofibroblasts (pdMFs) and in peripheral blood mononuclear cells (PBMCs). Effect of IL-24 on H 2 O 2 treated FHs74Int cells and on pdMFs was measured by MTT, LDH, Annexin V assays, real-time RT-PCR and by fluorescent microscopy. Results: We found increased level of IL-24 (3.3×, p < 0.05), α-SMA (2.4×, p < 0.05) and FN (2.3×, p < 0.05) in the duodenal mucosa and increased expression of IL19 (3.6×, p < 0.05) and IL24 (5.2×, p < 0.05) in the PBMCs of children with CD compared to that of controls. IL-1β was a strong inducer of IL24 expression of FHs74Int cells (9.9×, p < 0.05), pdMFs (552.9×, p < 0.05) or PBMCs (17.2×, p < 0.05), as well. IL-24 treatment reduced the number of apoptotic cells (0.5×, p < 0.05) and decreased the expression of inflammatory factors, including IL1A, IL6 and TNF of H 2 O 2-treated FHs74Int cells. IL-24 decreased the proliferation (0.6×, p < 0.05) of PDGF-B treated pdMFs. Moreover, IL-24 treatment altered the morphology of pdMFs by influencing the size of the angles between stress fibers and the longitudinal axis of the cells (2.0×, p < 0.05) and the expression of cytoskeletal components, including ACTA2, ACTB, VIM, SNAI1 and SNAI2. Conclusion: Our results suggest that IL-24 plays a significant role in the maintenance of duodenal mucosal integrity in CD.

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“…For the cardiac tissue of rats submitted to intermittent hypoxia, GAPDH was the most stable gene, followed by ACTB (actin β) (Julian et al, 2016). In the present study, the ACTB gene was not a candidate for reference gene because, according to Veres-Székely et al (2017), this gene should not be used as a reference gene when fibrosis can occur, because it generates cross-reactions of the expression of the differents actin isoforms, influencing its gene expression.…”

Section: Discussionmentioning

confidence: 98%

Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes

Oliveira

Livramento

Magalhães

et al. 2020

RSD

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The validation of reference genes is an essential step for any RT-qPCR analysis. In this way, the present paper aimed to identify and validate reference genes for RT-qPCR in cardiac tissue of rats of the Rattus norvegicus albinus specie, submitted to obesity associated or not to type 2 diabetes mellitus. For this, the metabolic changes were induced at the 42nd day of life and the euthanasia was performed on the 70th day. The heart apexes were collected and destinates for RNA extraction. The RT-qPCR technique was performed in own thermocycler, the efficiency of the primers found by the LinReg software and the stability of the expression of the reference genes in the samples was analyzed by the RefFinder algorithm. The candidates for reference genes were GAPDH, POLR2A, RPL32, and RPL4 and the target gene used to verify the differences in gene expression of candidates for reference genes was CMA1. The obese animals showed a decrease in CMA1 gene expression when compared to the two most stable reference genes. The opposite occurs when it is compared to the two less stable reference genes. The GAPDH and POLR2A genes are the best to normalize the reactions with the samples in question. There is no universal reference gene for all situations, which requires systematic validation for each situation. The use of unvalidated reference genes may compromise the interpretation of the expression of the target genes, which would prevent the reflection of the actual situation.

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Selective measurement of α smooth muscle actin: why β-actin can not be used as a housekeeping gene when tissue fibrosis occurs

Cited by 35 publications

References 26 publications

Age-Dependent Chronic Lung Injury and Pulmonary Fibrosis following Single Exposure to Hydrochloric Acid

Age-Dependent Chronic Lung Injury and Pulmonary Fibrosis following Single Exposure to Hydrochloric Acid

Role of IL-24 in the mucosal remodeling of children with coeliac disease

Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes

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