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Related Concept Videos

Circadian Rhythms and Gene Regulation02:19

Circadian Rhythms and Gene Regulation

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The biological clock is involved in many aspects of regulating complex physiology in all animals. It was in 1935 when German zoologists, Hans Kalmus and Erwin Bünning, discovered the existence of circadian rhythm in Drosophila melanogaster. However, the internal molecular mechanisms behind the circadian clock remained a mystery until 1984, when Jeffrey C. Hall, Michael Rosbash, and Michael W. Young discovered the expression of the Per gene oscillating over a 24-hour cycle. In subsequent...
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Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures
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Reference Gene Optimization for Circadian Gene Expression Analysis in Human Adipose Tissue.

Jeremy M White1, Matthew J Piron2, Vittobai R Rangaraj1

  • 1Committee on Molecular Metabolism and Nutrition, University of Chicago, Chicago, Illinois.

Journal of Biological Rhythms
|November 1, 2019
PubMed
Summary

Identifying stable reference genes is crucial for studying circadian rhythms in obesity. A three-gene panel (YWHAZ, RPL13α, GAPDH) provides optimal normalization for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in adipose tissue.

Keywords:
circadianhuman adipose tissueqRT-PCRreference genesleeve gastrectomyweight loss surgery

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Area of Science:

  • Chronobiology and Metabolism
  • Molecular Biology and Gene Expression

Background:

  • Circadian rhythms regulate organismal physiology, and their disruption is linked to obesity, insulin resistance, and diabetes.
  • Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is essential for studying circadian gene expression but requires reliable reference gene normalization.
  • Identifying suitable reference genes is challenging across diverse biological systems, particularly in metabolically relevant tissues like adipose tissue.

Purpose of the Study:

  • To identify and validate suitable reference genes for accurate measurement of circadian gene expression in human adipose tissue.
  • To assess the suitability of candidate reference genes (18S rRNA, GAPDH, HPRT1, RPII, RPL13α, YWHAZ) using multiple analytical tools.
  • To determine the optimal reference gene set for analyzing circadian oscillations in adipose tissue from morbidly obese women.

Main Methods:

  • Adipose tissue biopsies (subcutaneous and mesenteric) were collected from premenopausal morbidly obese women before and after sleeve gastrectomy.
  • Ex vivo sterile cultures of adipose tissue were maintained with serial sample collection every 4 hours for approximately 36 hours.
  • Candidate reference genes were analyzed using three distinct analytical tools to assess their expression stability and suitability for normalization in qRT-PCR.

Main Results:

  • None of the tested individual genes (18S rRNA, GAPDH, HPRT1, RPII, RPL13α, YWHAZ) were deemed suitable as a single reference gene control.
  • The optimal reference gene set for normalizing circadian gene expression in this adipose tissue model was identified as a geometric average of three genes: YWHAZ, RPL13α, and GAPDH.
  • This validated three-gene panel demonstrated suitability for measuring oscillations in the expression of a known circadian clock element (Dbp).

Conclusions:

  • A single reference gene is insufficient for accurate circadian gene expression analysis in human adipose tissue.
  • The combination of YWHAZ, RPL13α, and GAPDH, geometrically averaged, provides a robust normalization strategy for qRT-PCR in this context.
  • The presented methodology can be applied to identify optimal reference gene panels in other biological systems for circadian research.