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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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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Validating the classics: Accurate reference gene panel for reliable RT-qPCR in Porifera.

Kseniia V Skorentseva1, Nikolai P Melnikov2,3, Alexander V Ereskovsky1,4

  • 1Laboratory of Morphogenesis Evolution, Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, Russia.

Plos One
|May 20, 2026
PubMed
Summary

Selecting stable reference genes for quantitative real-time PCR (RT-qPCR) in sponges is crucial. This study identifies the most stable genes for Leucosolenia corallorrhiza and demonstrates that reference gene stability is species-specific across Porifera.

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Area of Science:

  • Marine Biology
  • Molecular Biology
  • Genomics

Background:

  • Quantitative real-time PCR (RT-qPCR) is vital for gene expression analysis.
  • Accurate normalization in RT-qPCR relies on stable reference genes.
  • Non-model organisms like sponges present challenges due to variability and limited resources.

Purpose of the Study:

  • To identify and validate stable reference genes for RT-qPCR in the calcareous sponge Leucosolenia corallorrhiza.
  • To assess the cross-species applicability of reference genes in distinct sponge species.
  • To provide a validated panel of reference genes for Porifera research.

Main Methods:

  • Seven candidate housekeeping genes were selected based on transcriptomic and genomic data.
  • Expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder in L. corallorrhiza and three other sponge species.
  • Pairwise variation analysis was used to determine the optimal number of reference genes for normalization.

Main Results:

  • RPL13A, ACT1, and GAPDH were identified as the most stable reference genes in Leucosolenia corallorrhiza.
  • While similar gene subsets showed stability across species, no single gene was universally stable.
  • Using two to three validated reference genes is recommended for accurate normalization in sponge RT-qPCR studies.

Conclusions:

  • Reference gene stability in sponges is species-specific and cannot be reliably predicted by phylogenetic or ecological factors.
  • A robust panel of candidate reference genes is provided for Porifera, requiring species- and condition-specific validation.
  • The study highlights the need to consider technical challenges in non-model organism research.