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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Search for appropriate reference genes for quantitative reverse transcription PCR studies in somite, prosencephalon and heart of early mouse embryo

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Author(s):
Moyses-Oliveira, Mariana [1] ; Cabral, Victoria [1] ; Gigek, Carolina Oliveira [1] ; de Carvalho Correa, Debora Cabral [1] ; Di-Battista, Adriana [1] ; Stumpp, Taiza [2] ; Melaragno, Maria Isabel [1]
Total Authors: 7
Affiliation:
[1] Univ Fed Sao Paulo, Dept Morphol & Genet, Div Genet, Sao Paulo - Brazil
[2] Univ Fed Sao Paulo, Dept Morphol & Genet, Dev Biol Lab, Sao Paulo - Brazil
Total Affiliations: 2
Document type: Journal article
Source: Gene; v. 710, p. 148-155, AUG 20 2019.
Web of Science Citations: 0
Abstract

qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative Delta Cq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development. (AU)

FAPESP's process: 14/11572-8 - Chromosomal rearrangements and their relevance in the etiology of genetic disorders: cytogenomic and molecular investigation
Grantee:Maria Isabel de Souza Aranha Melaragno
Support Opportunities: Research Projects - Thematic Grants