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Optimizing an ethanol-based fixative for enhanced nucleic acid preservation in cervical samples using a central
Ghazal Sedaghat Shayegan1, Pouya Salehipour1, Ali Najafi1
1Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Plos One
|June 26, 2026
Summary
A new ethanol-based fixative effectively preserves DNA and RNA in cervical samples at room temperature, improving cervical cancer diagnosis and Human Papillomavirus (HPV) testing.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Gynecologic Oncology
Background:
- Accurate cervical cancer diagnosis relies on high-quality DNA and RNA from clinical samples.
- Existing preservation methods face challenges with RNA stability and safety.
- There is a need for improved preservation techniques for cervical samples.
Purpose of the Study:
- To develop an optimized ethanol-based fixative for preserving DNA and RNA in cervical samples.
- To evaluate the fixative's performance under ambient conditions.
- To enable simultaneous molecular and cytological analysis of cervical samples.
Main Methods:
- HeLa cells and clinical samples were fixed using ethanol-based solutions with polyhydric compounds and chelating agents.
- A central composite design (CCD) optimized fixative composition (pH, Sorbitol, PEG concentrations).
- Nucleic acid quality was assessed via gel electrophoresis, PCR, and qRT-PCR; cellular morphology via Papanicolaou staining; HPV genotyping via real-time PCR.
Main Results:
- An optimized fixative (40% ethanol, 4.3% Sorbitol, 1.2% PEG 8000, pH 5.6) was developed.
- This formula demonstrated superior DNA and RNA preservation compared to PreservCyt.
- High-integrity DNA and RNA were obtained, enabling successful HPV oncogene amplification, rRNA analysis, and HPV genotyping.
Conclusions:
- The novel ethanol-based fixative offers a cost-effective, environmentally friendly solution for nucleic acid and cellular morphology preservation.
- Its efficacy at ambient temperatures supports its use for Human Papillomavirus (HPV) testing and cervical cancer screening.
- This fixative is particularly beneficial for resource-limited settings.
