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Reference Gene Stability in Agrostemma githago Using Quantitative Real-Time PCR.

Monika Bielecka1, Bartosz Pencakowski1, Marta Stafiniak1

  • 1Department of Pharmaceutical Biology and Biotechnology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland.

International Journal of Molecular Sciences
|January 28, 2026
PubMed
Summary
This summary is machine-generated.

This study identifies the most stable reference genes for quantitative real-time PCR (qPCR) in Agrostemma githago. Histone H3 (H3) and eukaryotic translation initiation factor 5A1 (TIF5A1-2) are recommended for accurate gene expression normalization.

Keywords:
Agrostemma githagoRefFinderhousekeeping genesqRT-PCR normalizationreference-gene validation

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Biotechnology

Background:

  • Quantitative real-time PCR (qPCR) is crucial for gene expression analysis, requiring validated reference genes for accurate normalization.
  • Commonly used reference genes show variable expression across different plant tissues, developmental stages, and experimental conditions.
  • No validated reference genes are currently available for Agrostemma githago (corn cockle).

Purpose of the Study:

  • To identify and validate the most stable reference genes for gene expression studies in Agrostemma githago.
  • To facilitate research on natural product biosynthesis and specialized metabolism regulation in this species.

Main Methods:

  • Seven candidate housekeeping genes were selected based on literature and transcriptome data.
  • Quantitative real-time PCR (qRT-PCR) was used to measure transcript levels in 40 diverse Agrostemma githago samples.
  • Expression stability was assessed using the RefFinder platform, integrating geNorm, NormFinder, BestKeeper, and Δ-Ct methods.

Main Results:

  • Reference gene stability varied significantly depending on the specific organ, developmental stage, and experimental condition.
  • TIF5A1-2 and GAPDH showed the most stable expression under various in vitro conditions.
  • EF1α and H3 were superior across different organs of soil-grown plants.
  • Histone H3 (H3) and TIF5A1-2 were identified as the two most stable, universal reference genes for Agrostemma githago across all tested conditions.

Conclusions:

  • The study successfully identified and validated the most stable reference genes for Agrostemma githago.
  • H3 and TIF5A1-2 are recommended for accurate normalization in qPCR studies of Agrostemma githago.
  • These findings provide a foundation for future transcriptomic and functional studies in Agrostemma githago and related species.