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Effects of brain tissue section processing and storage time on gene expression.

Erteng Jia1, Ying Zhou1, Huajuan Shi1

  • 1State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 210096, China.

Analytica Chimica Acta
|December 7, 2020
PubMed
Summary
This summary is machine-generated.

Frozen section storage conditions significantly impact RNA integrity and sequencing results. Storing samples at -20°C and using cresyl violet staining can help preserve RNA quality for accurate transcriptome analysis.

Keywords:
Frozen sectionGene expressionRNA integrityTranscriptomic

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Sample pre-processing is critical for RNA sequencing (RNA-seq) data accuracy.
  • Optimal frozen section storage conditions for RNA integrity and sequencing outcomes remain under-investigated.
  • Understanding these conditions is vital for reliable single-cell and spatial transcriptome sequencing.

Purpose of the Study:

  • To investigate the effects of different frozen section storage temperatures and durations on RNA integrity.
  • To assess the impact of storage conditions on RNA sequencing results and gene expression.
  • To identify stable reference genes and evaluate the protective effect of cresyl violet staining on RNA.

Main Methods:

  • RNA was isolated from brain sections stored at room temperature (RT) and -20°C for various time points (0h to 6 months).
  • RNA integrity was assessed using RNA integrity number (RIN).
  • Differential gene expression analysis was performed, and reference gene stability was validated using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR).

Main Results:

  • Storage at room temperature significantly decreased RIN values over time, while -20°C storage had a lesser impact.
  • Cresyl violet staining showed minimal effect on RNA integrity.
  • Numerous differentially expressed genes (DEGs) were identified, with some gene expressions (e.g., Gpm6a, Calm1) correlating with RNA quality.
  • 18S ribosomal RNA was identified as the most stable reference gene in brain tissue.

Conclusions:

  • Storage temperature and duration critically influence frozen section RNA integrity and RNA-seq results.
  • Certain genes exhibit stable expression despite declining overall RNA integrity.
  • 1% cresyl violet staining can protect RNA integrity in brain tissue sections.