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Selecting stable reference genes is crucial for accurate gene expression analysis in Codonopsis pilosula. This study identified optimal reference gene combinations for different conditions, improving the reliability of RT-qPCR data.

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Genomics

Background:

  • Accurate relative gene expression analysis using RT-qPCR relies on stable reference genes for normalization.
  • Inappropriate reference gene selection can lead to erroneous target gene expression data.

Purpose of the Study:

  • To identify and validate stable reference genes for gene expression studies in Codonopsis pilosula under various conditions.
  • To establish reliable normalization strategies for RT-qPCR in C. pilosula.

Main Methods:

  • RNA-Seq data was used to select 15 candidate reference genes based on expression variability (CV, FC).
  • RT-qPCR was employed to assess the expression stability of candidate genes across different growth stages and stress treatments (cold, drought).
  • Gene expression stability was evaluated using multiple algorithms (ΔCt, geNorm, NormFinder, Bestkeeper) and validated with a target gene (UDGPase).

Main Results:

  • Fifteen candidate reference genes were selected from transcriptome data.
  • Expression stability analysis identified optimal reference gene combinations for specific conditions: PP2A59γ, CPY20-1, UBCE32, RPL5B, UBC18 (developmental stage); RPL5B, RPL13, PP2A59γ (cold stress); RPL13, PP2A59γ (drought stress).

Conclusions:

  • The study provides validated reference gene sets for accurate gene expression normalization in Codonopsis pilosula.
  • These findings lay the groundwork for reliable reference gene selection and facilitate further research into the metabolic mechanisms of C. pilosula.