Housekeeping Gene Stability in Human Mesenchymal Stem and Tendon Cells Exposed to Tenogenic Factors

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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The usefulness of competitive PCR: airway gene expression of IL-5, IL-4, IL-4δ2, IL-2, and IFNγ in asthma

Thorax ◽

10.1136/thx.56.7.541 ◽

2001 ◽

Vol 56 (7) ◽

pp. 541-548

Author(s):

E M Glare ◽

M Divjak ◽

M J Bailey ◽

E H Walters

Keyword(s):

Gene Expression ◽

Inhaled Corticosteroids ◽

In Situ Hybridisation ◽

Housekeeping Gene ◽

Cross Sectional Study ◽

Mrna Levels ◽

Competitive Pcr ◽

Cross Sectional ◽

Steroid Use ◽

Biopsy Specimens

BACKGROUNDAsthma has been described as an eosinophilic bronchitis driven by interleukin(IL)-4 and IL-5. The quantification of cytokine mRNA levels in airway samples has been confounded by housekeeping gene expression which differs between and within asthmatics and controls.METHODSThe usefulness of competitive reverse transcriptase-polymerase chain reaction (RT-PCR) that is independent of housekeeping gene expression for quantitating the mRNA for interferon (IFN)γ, IL-2, IL-5, IL-4 and its receptor antagonist encoding splicing variant IL-4δ2 was determined in a cross sectional study of 45 normal control subjects and 111 with asthma.RESULTSAtopic controls and atopic asthmatic subjects expressed more IL-5 than non-atopic controls (p<0.02) in bronchoalveolar lavage (BAL) cells, but not in biopsy specimens. IL-5 mRNA expression in BAL cells from asthmatic subjects using inhaled corticosteroids (ICS) was significantly lower than those not receiving ICS (p=0.04). IL-2 mRNA levels differed with steroid use in biopsy specimens but not in BAL cells. IFNγ, IL-4, and IL-4δ2 mRNA levels did not differ between any groups and were not affected by steroid use. IL-4 and IL-4δ2 mRNA levels were positively correlated (p<0.0001), suggesting coordinated transcription.CONCLUSIONSWhile the signal differentiation of competitive PCR in asthma may rival that of in situ hybridisation and immunohistochemistry, the method is expensive and wasteful of material.

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Assessment of the Telomere Length and Its Effect on the Symptomatology of Parkinson’s Disease

Antioxidants ◽

10.3390/antiox10010137 ◽

2021 ◽

Vol 10 (1) ◽

pp. 137

Author(s):

Tina Levstek ◽

Sara Redenšek ◽

Maja Trošt ◽

Vita Dolžan ◽

Katarina Trebušak Podkrajšek

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Telomere Length ◽

Repetitive Sequences ◽

Housekeeping Gene ◽

Cellular Aging ◽

Brain Diseases ◽

Motor Complications ◽

Telomere Attrition ◽

Significant Difference

Telomeres, which are repetitive sequences that cap the end of the chromosomes, shorten with each cell division. Besides cellular aging, there are several other factors that influence telomere length (TL), in particular, oxidative stress and inflammation, which play an important role in the pathogenesis of neurodegenerative brain diseases including Parkinson’s disease (PD). So far, the majority of studies have not demonstrated a significant difference in TL between PD patients and healthy individuals. However, studies investigating the effect of TL on the symptomatology and disease progression of PD are scarce, and thus, warranted. We analyzed TL of peripheral blood cells in a sample of 204 PD patients without concomitant autoimmune diseases and analyzed its association with several PD related phenotypes. Monochrome multiplex quantitative PCR (mmqPCR) was used to determine relative TL given as a ratio of the amount of DNA between the telomere and albumin as the housekeeping gene. We found a significant difference in the relative TL between PD patients with and without dementia, where shorter TL presented higher risk for dementia (p = 0.024). However, the correlation was not significant after adjustment for clinical factors (p = 0.509). We found no correlations between TLs and the dose of dopaminergic therapy when the analysis was adjusted for genetic variability in inflammatory or oxidative factors. In addition, TL influenced time to onset of motor complications after levodopa treatment initiation (p = 0.0134), but the association did not remain significant after adjustment for age at inclusion and disease duration (p = 0.0781). Based on the results of our study we conclude that TL contributes to certain PD-related phenotypes, although it may not have a major role in directing the course of the disease. Nevertheless, this expends currently limited knowledge regarding the association of the telomere attrition and the disease severity or motor complications in Parkinson’s disease.

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Stress-deprivation induces an up-regulation of versican and connexin-43 mRNA and protein synthesis and increased ADAMTS-1 production in tendon cells in situ

Connective Tissue Research ◽

10.1080/03008207.2021.1873302 ◽

2021 ◽

pp. 1-10

Author(s):

Monika Egerbacher ◽

Keri Gardner ◽

Oscar Caballero ◽

Juraj Hlavaty ◽

Sarah Schlosser ◽

...

Keyword(s):

Protein Synthesis ◽

Connexin 43 ◽

Tendon Cells

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Magnetic responsive materials modulate the inflammatory profile of IL-1β conditioned tendon cells

Acta Biomaterialia ◽

10.1016/j.actbio.2020.09.028 ◽

2020 ◽

Vol 117 ◽

pp. 235-245

Author(s):

A. Vinhas ◽

M.T. Rodrigues ◽

A.I. Gonçalves ◽

R.L. Reis ◽

M.E. Gomes

Keyword(s):

Responsive Materials ◽

Tendon Cells ◽

Inflammatory Profile

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Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

Microbiology ◽

10.1099/mic.0.038224-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2080-2091 ◽

Cited By ~ 28

Author(s):

Anne-Laure Michon ◽

Fabien Aujoulat ◽

Laurent Roudière ◽

Olivier Soulier ◽

Isabelle Zorgniotti ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Diversity ◽

Natural Populations ◽

Variable Region ◽

Housekeeping Gene ◽

Single Copy ◽

Population Based ◽

Rrna Gene ◽

Gene Copies

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

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Oxidative energy metabolism in equine tendon cells

Research in Veterinary Science ◽

10.1016/s0034-5288(97)90127-2 ◽

1997 ◽

Vol 62 (2) ◽

pp. 93-97 ◽

Cited By ~ 24

Author(s):

H.L Birch ◽

G.A Rutter ◽

A.E Goodship

Keyword(s):

Energy Metabolism ◽

Oxidative Energy Metabolism ◽

Tendon Cells

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