Improvement in RNA quantity and quality in cervico-vaginal cytology

Gun Oh. Chong,Yoon Hee Lee,Hyung Soo Han,Seon Duk Lee

doi:10.1186/s12985-020-1282-x

Gun Oh. Chong, Yoon Hee Lee + Show 2 more

Open Access

https://doi.org/10.1186/s12985-020-1282-x

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Abstract

The separation of exfoliated cells from the brushes used during cervico-vaginal smears is difficult, a problem which may affect the quality of ribonucleic acid (RNA) extracted. We compared the results of RNA extraction from cervico-vaginal cytology samples according to the type of tubes, preservative solutions, and storage temperature. The samples included exfoliated cervico-vaginal cytological specimens from patients with human papilloma virus 16, positive for cervical intraepithelial neoplasia or cervical cancer. Exfoliated cells were obtained by shaking a brush in a conventional rigid vial tube or squeezing the brush in a soft vial tube. RNA quantity and quality were compared between the two tubes. The concentration and purity of RNA (A260/A280 and A260/A230 ratios) was compared amongst five groups: Group 1, standard frozen storage; Group 2–4, RNA stabilization reagents with room temperature [RNAlater RNA Stabilization Reagent, RNAprotect cell Reagent and AllProtect Tissue Reagent]; and Group 5, Surepath Preservative fluid. To demonstrate the utility of the extracted RNA for PCR-based cDNA synthesis, GAPDH and E6 were targeted and gel band densities of GAPDH and E6 were measured. The median RNA concentration was significantly higher in the soft tubes compared with the rigid tubes (100.2 vs. 7.1 ng/μL, p = 0.0209). The purity of the RNA was higher in soft vial tubes than in rigid vials, as measured by A260/280 and A260/230 ratios. The RNA concentration, purity, and GAPDH density of groups 1, 2 and 3 were significantly higher than those of groups 4 and 5. Moreover, E6 density of group 1 and 2 was significantly higher than that of group 3, 4 and 5. The use of soft tubes enhanced the mRNA quantity and quality in cervico-vaginal cytology. The products of mRNA extraction using RNAlater RNA Stabilization Reagent and RNAprotect Cell Reagent at room temperature were comparable to those obtained by conventional frozen storage. Our protocol improved the yield and quality of RNA and might produce better results for molecular analysis in cervico-vaginal cytology.

Highlights

Exfoliated cytology has been proposed as a method which can provide information about epithelial cells such as screening for precancerous lesions or ribonucleic acid (RNA) expression [1]
The products of messenger Riboucleic Acid (mRNA) extraction using RNAlater RNA Stabilization Reagent and RNAprotect Cell Reagent at room temperature were comparable to those obtained by conventional frozen storage
The aim of this study was 1) to compare methods of RNA extraction of cervico-vaginal cytology as they relate to the type of tubes, preservative solutions, and storage temperature and 2) to establish the best technique for molecular tests such as real-time reverse transcriptase reaction or RNA-seq at room temperature

Summary

Introduction

Exfoliated cytology has been proposed as a method which can provide information about epithelial cells such as screening for precancerous lesions or RNA expression [1]. The use of soft tubes enhanced the mRNA quantity and quality in cervico-vaginal cytology. The products of mRNA extraction using RNAlater RNA Stabilization Reagent and RNAprotect Cell Reagent at room temperature were comparable to those obtained by conventional frozen storage.

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