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Related Concept Videos

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Related Experiment Video

Updated: Jan 6, 2026

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere
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Quantifying Soil Microbiome Abundance by Metatranscriptomics and Complementary Molecular Techniques-Cross-Validation

Mathilde Borg Dahl1, Stella Brachmann1, Andrea Söllinger2

  • 1Department of Bacterial Physiology, University Greifswald, Greifswald, Germany.

Molecular Ecology Resources
|June 3, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces an internal RNA standard for soil metatranscriptomics, aiming to quantify microbial biomass. While the standard aids RNA extraction assessment, biomass estimates varied significantly, highlighting challenges in soil nucleic acid quantification.

Keywords:
RNAbiomass estimatesextraction standardmetatranscriptomicsquantitative transcriptomics

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Area of Science:

  • Soil microbiology
  • Molecular ecology
  • Biogeochemistry

Background:

  • Linking soil microbial community structure and function to biogeochemical processes is crucial but methodologically challenging.
  • Quantitative soil metatranscriptomics requires accurate estimation of microbial biomass.
  • Internal standards can improve the reliability of nucleic acid extraction and quantification.

Purpose of the Study:

  • To evaluate an internal RNA extraction standard (NAEstd) for quantitative microbial biomass estimation in soil metatranscriptomics.
  • To compare biomass estimates derived from NAEstd, qRT-PCR, and a previous method (qMeTra) with soil microbial biomass carbon (MBC) and nitrogen (MBN).
  • To assess the impact of laboratory protocols and soil properties on RNA extraction efficiency and biomass quantification.

Main Methods:

  • Metatranscriptomic sequencing of soil samples using two library preparation protocols in triplicates.
  • Addition of an internal RNA extraction standard (NAEstd) from *Saccharolobus solfataricus* to soil samples.
  • Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for 16S rRNA transcript abundance.
  • Comparison of biomass estimates with microbial biomass carbon (MBC) and nitrogen (MBN) measurements.

Main Results:

  • High accuracy (99.9%) in identifying NAEstd reads and good replication consistency between triplicates (Bray-Curtis dissimilarity 0.03).
  • A clear bias was observed between library preparation protocols, although it did not affect the between-sample patterns.
  • Biomass estimates derived from different methods (NAEstd, qRT-PCR, qMeTra) varied by orders of magnitude, with no correlation between NAEstd and MBC.
  • Significant correlations were found between most biomass estimation methods, except for NAEstd and MBC.

Conclusions:

  • The internal RNA standard (NAEstd) shows potential for assessing nucleic acid retention during soil extraction.
  • Accurate microbial biomass estimation in soil metatranscriptomics remains challenging due to variations in community size and soil nucleic acid retention.
  • Further research is needed to refine methods for robust quantitative biomass estimation in complex soil environments.