Optimizing an ethanol-based fixative for enhanced nucleic acid preservation in cervical samples using a central composite design approach

Sep 23, 2026

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High-quality DNA and RNA from cervical specimens are essential for accurate cervical cancer diagnosis, yet commonly used preservation approaches can compromise RNA stability and raise safety concerns. This study develops an optimized ethanol-based fixative for ambient-condition storage. HeLa cells and clinical samples were evaluated using a central composite design, varying pH, sorbitol, and PEG 8000. Nucleic acid integrity was assessed by gel electrophoresis, PCR, qRT-PCR, and HPV genotyping, alongside Papanicolaou-based morphology.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages20

Reading Time50 min

AccessFree to download

Abstract

Background

Methods

Results

Conclusion

Introduction

Cervical cancer and HPV risk

Molecular diagnostics and the need for nucleic acid integrity

Approved HPV diagnostic methods and sample requirements

Limitations of morphology and DNA-only screening

OPEN ACCESS Citation: Sedaghat Shayegan G, Salehipour P, Najafi A, Modarressi MH (2026) Optimizing an ethanol-based fixative for enhanced nucleic acid preservation in cervical samples using a central composite design approach. PLoS One 21(6): e0349088. [https://doi.org/10.1371/journal](https://doi.org/10.1371/journal). pone.0349088 Editor: Devesh U Kapoor, Dr. Dayaram Patel Pharmacy College, INDIA Received: July 4, 2025 Accepted: April 23, 2026 Published: June 26, 2026 Copyright: © 2026 Sedaghat Shayegan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data availability statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by the Tehran University of Medical Sciences, under RESEARCH ARTICLE Optimizing an ethanol-based fixative for enhanced nucleic acid preservation in cervical samples using a central composite design approach Ghazal Sedaghat Shayegan1, Pouya Salehipour1, Ali Najafi1, Mohammad Hossein Modarressi1,2* 1 Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 2 Genomics Department, Nilou Medical Laboratory, Tehran, Iran * [Modaresi@tums.ac.ir](Modaresi@tums.ac.ir) Abstract Background To accurately diagnose cervical cancer, high-quality genetic material (DNA and RNA) from clinical samples is crucial. Current preservation methods often have limitations, including poor RNA stability and safety concerns. This study aimed to develop an optimized ethanol-based fixative to preserve DNA and RNA in cervical samples under ambient conditions. Methods HeLa cells were fixed in ethanol-based fixatives containing polyhydric compounds (Sorbitol and polyethylene glycol [PEG]) and chelating agents. We used a central composite design (CCD) approach to evaluate the effects of pH, Sorbitol concentration, and PEG concentration on nucleic acid preservation. DNA and RNA quality were assessed using agarose gel electrophoresis, PCR, and real-time PCR. Cellular morphology was evaluated using Papanicolaou-stained slides. HPV genotyping of clinical samples was conducted using real-time PCR. Results The optimized fixative, developed in this study consisted of 40% ethanol, 4.3% Sorbitol, 1.2% PEG 8000, with a pH of 5.6. This new formula significantly improved DNA and RNA preservation compared to the commercial solution, PreservCyt. DNA showed high integrity and was successfully amplified in PCR test targeting HPV-18 oncogenes. RNA quality was confirmed through clear 28S and 18S rRNA bands and lower threshold cycle (Ct) values in qRT-PCR. HPV genotyping and morphological PLOS One | [https://doi.org/10.1371/journal.pone.0349088](https://doi.org/10.1371/journal.pone.0349088) June 26, 2026 1 / 20 Grant number 1400-1-410-52883. The fundershad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. analysis revealed excellent preservation, enabling both molecular and cytological evaluations. Conclusion The new ethanol-based fixative represents a promising cost-effective and environmentally friendly solution for preserving nucleic acids and cellular morphology in cervical samples. Its good performance under ambient conditions suggests it may serve as an option for Human Papillomavirus (HPV) testing and cervical cancer prevention, particularly in places with limited resources. 1. Introduction Cervical cancer is an important global health issue that particularly affects the female reproductive system. Cervical cancer is the fourth most common cancer in women worldwide, according to the World Health Organization (WHO) scale [ 1] . Human papillomavirus (H

FAQs

What is the main goal of the study on cervical samples?

To optimize an ethanol-based fixative that preserves DNA and RNA from cervical specimens stored under ambient conditions for reliable molecular and cytological testing.

Which experimental factors were optimized using the central composite design?

pH, Sorbitol concentration, and PEG concentration, assessed for their effects on nucleic acid preservation quality.

How did the optimized fixative perform compared with the commercial PreservCyt solution?

It significantly improved DNA and RNA preservation, showing high DNA integrity with successful PCR amplification (including HPV-18 oncogene targeting) and clear rRNA bands with lower qRT-PCR Ct values.

Sep 23, 2026

Uploaded by Aldword