QRT-PCR analysis of the effect of in utero exposure to sewage sludge on steroidogenic gene expression in ovine foetal adrenal gland

Abstract Introduction “Tuft” cells, also known as brush or caveolated cells, are characteristically fusiform shaped, with a distinct apical “tuft” of microvilli extending into the lumen. Double cortin-like kinase 1 (DCLK1) is a microtubule kinase and is a specific marker of intestinal tuft cells. DCLK1-positive tuft cells have been shown to play a key role in gastrointestinal chemosensation, inflammation, and neurotransmission. DCLK1 and Choline acetyltransferase (ChAT), the enzymes responsible for acetylcholine production, are reported to be coexpressed within the gastrointestinal tract. We designed this study to investigate the hypothesis that DCLK1 gene expression is altered in Hirschsprung's disease (HSCR). Materials and Methods HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was undertaken to quantify DCLK1 gene expression, and immunolabeling of DCLK1-positive tuft cells was visualized using confocal microscopy. Results qRT-PCR analysis revealed significant downregulation of the DCLK1 gene in both aganglionic and ganglionic HSCR specimens compared with controls (p < 0.05). Confocal microscopy revealed DCLK1-positive tuft cell expression within the colonic mucosa, with a reduction in expression in both aganglionic and ganglionic HSCR colon compared with controls. Conclusion DCLK1 is significantly downregulated in HSCR colon, suggesting a role for tuft cells in cholinergic neurotransmission of the distal colon. The marked decrease in DCLK1 expression within ganglionic specimens highlights the physiologically abnormal nature of this segment in HSCR patients.

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Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia

International Journal of Molecular Sciences ◽

10.3390/ijms20010034 ◽

2018 ◽

Vol 20 (1) ◽

pp. 34 ◽

Cited By ~ 3

Author(s):

Jing-Jing Wang ◽

Shuo Han ◽

Weilun Yin ◽

Xinli Xia ◽

Chao Liu

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Elongation Factor ◽

Pcr Analysis ◽

Qrt Pcr ◽

Gene Normalization ◽

Accurate Normalization ◽

Suitable Reference ◽

Different Tissues ◽

Gene Expression Levels

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

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Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera

Scientific Reports ◽

10.1038/s41598-020-70965-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ji Hyang Jeon ◽

KyungHwan Moon ◽

YeongHo Kim ◽

Young Ho Kim

Keyword(s):

Gene Expression ◽

Apis Mellifera ◽

Honey Bee ◽

Gene Selection ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Pcr Analysis ◽

Specific Gene ◽

Qrt Pcr ◽

Selection For

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Alteration of Mammary Gland Development and Gene Expression by In Utero Exposure to Cadmium

International Journal of Molecular Sciences ◽

10.3390/ijms18091939 ◽

2017 ◽

Vol 18 (9) ◽

pp. 1939 ◽

Cited By ~ 8

Author(s):

Daniela Parodi ◽

Morgan Greenfield ◽

Claire Evans ◽

Anna Chichura ◽

Alexandra Alpaugh ◽

...

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Mammary Gland Development ◽

In Utero ◽

In Utero Exposure ◽

Gland Development

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Comparison of Stemness and Gene Expression between Gingiva and Dental Follicles in Children

Stem Cells International ◽

10.1155/2016/8596520 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Chung-Min Kang ◽

Seong-Oh Kim ◽

Mijeong Jeon ◽

Hyung-Jun Choi ◽

Han-Sung Jung ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Pcr Analysis ◽

Expression Levels ◽

Qrt Pcr ◽

Stem Cell Source ◽

Regenerative Dentistry ◽

Induced Pluripotent

The aim of this study was to compare the differential gene expression and stemness in the human gingiva and dental follicles (DFs) according to their biological characteristics. Gingiva (n=9) and DFs (n=9) were collected from 18 children. Comparative gene expression profiles were collected using cDNA microarray. The expression of development, chemotaxis, mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSs) related genes was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Histological analysis was performed using hematoxylin-eosin and immunohistochemical staining. Gingiva had greater expression of genes related to keratinization, ectodermal development, and chemotaxis whereas DFs exhibited higher expression levels of genes related to tooth and embryo development. qRT-PCR analysis showed that the expression levels of iPSc factors includingSOX2,KLF4, andC-MYCwere58.5±26.3,12.4±3.5, and12.2±1.9times higher in gingiva andVCAM1(CD146) andALCAM(CD166) were33.5±6.9and4.3±0.8times higher in DFs. Genes related to MSCs markers includingCD13,CD34,CD73,CD90, andCD105were expressed at higher levels in DFs. The results of qRT-PCR and IHC staining supported the microarray analysis results. Interestingly, this study demonstrated transcription factors of iPS cells were expressed at higher levels in the gingiva. Given the minimal surgical discomfort and simple accessibility, gingiva is a good candidate stem cell source in regenerative dentistry.

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