Quantification of Transcript-to-Template Ratios as a Measure of Gene Expression Using RT-PCR

Harting, Inga; Wiesner, Rudolf J.

doi:10.2144/97233st01

Cited by 8 publications

(3 citation statements)

References 7 publications

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“…RNA isolation and mRNA analysis by semiquantitative RT-PCR To assess the level of gene expression of gill Na C ,K C -ATPase a-subunit and CFTR in striped bass, a semi-quantitative reverse transcription PCR (RT-PCR) approach was used (Harting & Wiesner 1997). Total RNA was isolated from gill tissues fixed in RNAlater using the TRIzol procedure (Invitrogen).…”

Section: Plasma [Namentioning

confidence: 99%

Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+-ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

Madsen

Jensen

Tipsmark

et al. 2007

Journal of Endocrinology

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Effects of salinity and hormones on cystic fibrosis transmembrane conductance regulator (CFTR) and α-subunit Na+,K+-ATPase (α-NKA) mRNA (analysed by semi-quantitative PCR) and protein expression (analysed by western blotting and immunocytochemistry) were investigated in gills of striped bass. Freshwater (FW) to seawater (SW) transfer induced a disturbance in serum [Na+]. Gill CFTR protein, mRNA level and Na+,K+-ATPase activity were unaffected by SW transfer, whereas α-NKA mRNA increased after transfer. CFTR immunoreactivity was observed in large cells in FW and SW gill filaments at equal intensity. Cortisol decreased serum [Na+] in FW fish, but had no effect on gill Na+,K+-ATPase activity, α-NKA and CFTR mRNA levels. Incubation of gill tissue with cortisol (24 h, >0.01 μg/ml) and epidermal growth factor (EGF 10 μg/ml) decreased CFTR mRNA levels relative to pre-incubation and control levels. CFTR expression was unaffected by IGF-I (10 μg/ml). α-NKA mRNA levels decreased by 50% after 24 h control incubation; it was slightly stimulated by cortisol and unaffected by IGF-I and EGF. In isolated gill cells, phosphorylation of extracellular-regulated kinase (ERK) 1/2 was stimulated by EGF but not affected by IGF-I. This study is the first to report a branchial EGF response and to demonstrate a functional ERK 1/2 pathway in the teleost gill. In conclusion, CFTR and Na+,K+-ATPase are differentially regulated by salinity and hormones in gills of striped bass, despite the putative involvement of both in salt excretion.

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Section: Plasma [Namentioning

confidence: 99%

Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+-ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

Madsen

Jensen

Tipsmark

et al. 2007

Journal of Endocrinology

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“…These products can be encoded either in the nucleus or in the mitochondria themselves. To study AmIF-2mt mRNA levels during critical queen versus worker developmental stages in more detail, we performed a semiquantitative RT-PCR analysis (Harting and Wiesner, 1997). Total RNA samples from queens and workers of fourth-instar and fifth-instar larvae, carefully classified by age and mass, were used as templates (see Materials and methods).…”

Section: Amif-2mt Mrna Levels Are Differentially Expressed In Queenmentioning

confidence: 99%

“…Higher mitochondrial activity can be achieved among other possibilities by increasing mitochondrial protein synthesis. In animal mitochondria, the molar concentration of all the mitochondrial mRNAs exceeds by far the concentration of ribosomes, a situation that is different from that in the cytosolic compartment (Garstka et al, 1994;Harting and Wiesner, 1997). Since the rate of translation initiation depends on mRNA and ribosome concentrations (Xia, 1996), the translation initiation factor may be a key regulator for mitochondria protein synthesis.…”

Section: Differences Between Queens and Workers In The Expression Ofmentioning

confidence: 99%

Differential expression of mitochondrial genes between queens and workers during caste determination in the honeybeeApis mellifera

Corona

Estrada

Zurita

1999

Journal of Experimental Biology

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The nourishment received by female honeybee larvae determines their differentiation into queens or workers. In this study, we report the first molecular analysis of differences that occur between queens and workers during the caste-determination process. RNA-differential display experiments identified a clone that encodes for a gene that is homologous to the nuclear-encoded mitochondrial translation initiation factor (AmIF-2mt). Semi-quantitative analysis by reverse transcriptase/polymerase chain reaction (RT-PCR) throughout honeybee development detected a higher level of expression of this gene in queen larvae than in worker larvae. Analysis of two other genes encoding mitochondrial proteins, cytochrome oxidase subunit 1 (COX-1; mitochondrial-encoded) and cytochrome c (cyt c; nuclear-encoded) also showed differential expression of these two genes between queens and workers. In particular, the cyt c transcript is more abundant in queen larvae and throughout the metamorphosis of the queen. These results indicate that the higher respiratory rate previously documented in queen larvae is accomplished through a higher level of expression of both nuclear- and mitochondrial-encoded genes for mitochondrial proteins.

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Interactive effects of cortisol treatment and ambient seawater challenge on gill Na+,K+-ATPase and CFTR expression in two strains of Atlantic salmon smolts

et al. 2003

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Quantification of Transcript-to-Template Ratios as a Measure of Gene Expression Using RT-PCR

Abstract: ABSTRACT

Cited by 8 publications

References 7 publications

Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+-ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+-ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

Differential expression of mitochondrial genes between queens and workers during caste determination in the honeybeeApis mellifera

Interactive effects of cortisol treatment and ambient seawater challenge on gill Na+,K+-ATPase and CFTR expression in two strains of Atlantic salmon smolts

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