Improvement in RNA quantity and quality in cervico-vaginal cytology

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 cervicovaginal 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.

Development and Validation of Rapid Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification for Detection of Rift Valley Fever Virus

“…The quality and integrity of the template used in the development of any diagnostic kit are as good as the kit itself. Therefore, the emphasis on reliance on a high-quality template cannot be underestimated [ 34 ]. In this study, total RNA was extracted from Vero cells infected with RVFV showing high levels of CPE ( Figure 2 ).…”

Optimization of pre‐analytical and analytical steps for DNA and RNA analysis of fresh cytology samples

“…Stability of nucleic acids in cytological samples was according to our knowledge studied by one research group. Chong et al 7 did a study about RNA extraction methods for cervico‐vaginal cytology samples according to tube types, storage temperatures, and preservative solutions. However, Chong et al did a comparison between samples stored at room temperature and freezer temperature, so our results cannot be compared, since the samples were in a preservative solution containing chemical for nucleic acid stabilization.…”

“…While pre‐analytical steps for molecular tests on formalin‐fixed paraffin‐embedded (FFPE) tissue samples are relatively standardized, the variety of cytological sample preparation techniques poses a challenge for standardized molecular testing. Numerous studies already compared different extraction methods of nucleic acids from various cytological samples, 3–5 however there is limited data for extraction of nucleic acids from fresh unfixed cell samples 6–8 …”

“…Numerous studies already compared different extraction methods of nucleic acids from various cytological samples, [3][4][5] however there is limited data for extraction of nucleic acids from fresh unfixed cell samples. [6][7][8] Different cytological preparations have already been described for use in molecular testing: smears, cell blocks, cytospins, and liquid-based cytology (LBC) samples, each with its advantages and disadvantages for molecular testing. 9,10 Smears are readily prepared and enable rapid onsite evaluation (ROSE) of the sample.…”

“…Numerous studies already compared different extraction methods of nucleic acids from various cytological samples, 3 , 4 , 5 however there is limited data for extraction of nucleic acids from fresh unfixed cell samples. 6 , 7 , 8 …”