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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Related Experiment Video

Updated: Feb 22, 2026

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis
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Optimal reference genes for RT-qPCR normalization in the newborn.

P Khanna1, K L Johnson2, J L Maron3

  • 1a Sackler School of Graduate Biomedical Sciences.

Biotechnic & Histochemistry : Official Publication of the Biological Stain Commission
|September 15, 2017
PubMed
Summary
This summary is machine-generated.

Identifying stable reference genes for newborn gene expression analysis is crucial. GAPDH and YWHAZ are reliable in neonatal saliva, unlike ACTB, which shows significant variation, especially in males.

Keywords:
ACTBGAPDHRT-qPCRYWHAZneonatalnormalizationreference genessaliva

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Area of Science:

  • Neonatal transcriptomics
  • Molecular biology
  • Biomarker discovery

Background:

  • Accurate gene expression analysis in newborns requires reliable reference genes.
  • Rapid neonatal development causes dynamic gene expression changes, complicating reference gene selection.
  • Stable reference genes are essential for studying neonatal morbidities like retinopathy of prematurity using RT-qPCR.

Purpose of the Study:

  • To evaluate the stability of commonly used reference genes (ACTB, GAPDH, YWHAZ) in neonatal saliva.
  • To identify optimal reference genes for quantitative gene expression studies in neonates across various gestational ages and sexes.
  • To assess potential sex-specific differences in reference gene expression in newborns.

Main Methods:

  • Total RNA extraction from 400 neonatal saliva samples (32 5/7 to 48 2/7 weeks postconceptional age).
  • cDNA conversion, pre-amplification, and quantitative PCR (qPCR) analysis of ACTB, GAPDH, and YWHAZ.
  • Relative quantification using the Δ Ct method, with data analyzed overall and stratified by age and sex.

Main Results:

  • ACTB exhibited significant expression variation across all neonates.
  • GAPDH and YWHAZ demonstrated greater stability compared to ACTB.
  • ACTB showed increased expression variation in males versus females, while GAPDH and YWHAZ remained stable across sexes.

Conclusions:

  • ACTB is an unreliable reference gene for neonatal transcriptomic studies.
  • GAPDH and YWHAZ are recommended as stable reference genes for RT-qPCR in neonates.
  • The findings improve reference gene selection for accurate gene expression analysis in rapidly developing newborns.