Optimization of reference gene panels for gene expression analysis in preclinical models of inflammatory skin diseases

Braun, Anne; Sezin, Tanya; Bezdek, Siegfried; Doxastaki, Iosifina; Zillikens, Detlef; Busch, Hauke; Sadik, Christian D.

doi:10.1111/exd.13989

Cited by 3 publications

(3 citation statements)

References 6 publications

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“…It allows to define minimal number of reference genes for accurate normalization. Cut-off threshold 0.15 is recommended to find out the optimal number reference genes (27,39). In our test, all pairwise variations in both total RNA and mRNA cases were below 0.15 (Fig.…”

Section: Expression Stability Analysis and Determination Of Minimal Number Of Reference Genes For Normalizationmentioning

confidence: 78%

A method to quantitate maternal transcripts localized in sea urchin egg cortex by RT-qPCR with accurate normalization

Yakovlev

Kipryushina

Maiorova

2021

Preprint

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The sea urchin egg cortex is a peripheral region of eggs consisting of cell membrane and adjacent cytoplasm, which contains actin and tubulin cytoskeleton, cortical granules and some proteins required for early development. Method for isolation of cortices from sea urchin eggs and early embryos has been developed in 70s of 20th Century. Since that time this method has been reliable tool to study protein localization and cytoskeletal organization in cortex of unfertilized eggs and embryos during first cleavages. This study is an estimation of reliability of RT-qPCR to analyze levels of maternal transcripts that are localized in egg cortex. Firstly, we selected seven potential reference genes, 28S, Cycb, Ebr1, GAPDH, Hmg1, Smtnl1 and Ubb, which transcripts are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, ΔCt, geNorm and NormFinder) upon calculated level stability in both eggs and isolated cortices. Our results show that gene ranking differs in total RNA and mRNA samples, though Ubb is most suitable reference gene in both cases. To validate feasibility of comparative analysis of eggs and isolated egg cortices by RT-qPCR, we selected Daglb-2 as a gene of interest, which transcripts potentially localized in cortex, and found increased level of Daglb-2 in egg cortices. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to approve cortical association of transcripts in sea urchin eggs.

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Section: Expression Stability Analysis and Determination Of Minimal Number Of Reference Genes For Normalizationmentioning

confidence: 78%

A method to quantitate maternal transcripts localized in sea urchin egg cortex by RT-qPCR with accurate normalization

Yakovlev

Kipryushina

Maiorova

2021

Preprint

View full text Add to dashboard Cite

show abstract

“…It allows defining minimal number of reference genes for accurate normalization. Cut-off threshold of 0.15 is recommended to determine the optimal number reference genes [ 27 , 44 ]. In our test, all pairwise variations in both total RNA and mRNA cases revealed M value <0.15 ( Fig 4 ), which point to usage of two reference genes for normalization.…”

Section: Resultsmentioning

confidence: 99%

An approach to quantitate maternal transcripts localized in sea urchin egg cortex using RT-qPCR with accurate normalization

2022

View full text Add to dashboard Cite

The sea urchin egg cortex is a peripheral region of eggs comprising a cell membrane and adjacent cytoplasm, which contains actin and tubulin cytoskeleton, cortical granules and some proteins required for early development. Method for isolation of cortices from sea urchin eggs and early embryos was developed in 1970s. Since then, this method has been reliable tool to study protein localization and cytoskeletal organization in cortex of unfertilized eggs and embryos during first cleavages. This study was aimed to estimate the reliability of RT-qPCR to analyze levels of maternal transcripts that are localized in egg cortex. Firstly, we selected seven potential reference genes, 28S, Cycb, Ebr1, GAPDH, Hmg1, Smtnl1 and Ubb, the transcripts of which are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, ΔCt, geNorm and NormFinder) based on calculated level of stability in both eggs as well as isolated cortices. Our results showed that gene ranking differs in total RNA and mRNA samples, though Ubb is most suitable reference gene in both cases. To validate feasibility of comparative analysis of eggs and isolated egg cortices, we selected Daglb-2 as a gene of interest, which transcripts are potentially localized in cortex according to transcriptome analysis, and observed increased level of Daglb-2 in egg cortices by RT-qPCR. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to determine cortical association of transcripts in sea urchin eggs.

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“…Im Laufe der Jahre hat sich so eine sehr fruchtbare wissenschaftliche Interaktion herausgebildet mit zahlreichen Forschungsaufenthalten in beiden Ländern. Eine Auswahl daraus entstandener Veröffentlichungen ist in der Literatur widergegeben .…”

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Überwältigende Gastfreundschaft der Israelischen Dermatologischen Gesellschaft anlässlich der 40. dermatologischen Jahrestagung in Eilat unter dem Motto: GET C‐O‐N‐N‐E‐C‐T‐E‐D

Lehmann

2019

J Deutsche Derma Gesell

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Optimization of reference gene panels for gene expression analysis in preclinical models of inflammatory skin diseases

Cited by 3 publications

References 6 publications

A method to quantitate maternal transcripts localized in sea urchin egg cortex by RT-qPCR with accurate normalization

A method to quantitate maternal transcripts localized in sea urchin egg cortex by RT-qPCR with accurate normalization

An approach to quantitate maternal transcripts localized in sea urchin egg cortex using RT-qPCR with accurate normalization

Überwältigende Gastfreundschaft der Israelischen Dermatologischen Gesellschaft anlässlich der 40. dermatologischen Jahrestagung in Eilat unter dem Motto: GET C‐O‐N‐N‐E‐C‐T‐E‐D

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