Evaluation of two RNA extraction methods in children's saliva

Anaya, Meisser Vidal Madera; Causado, Amileth Suárez

doi:10.1016/j.rodmex.2018.01.014

Cited by 11 publications

(9 citation statements)

References 29 publications

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“…We achieved an average value for A260/280 ratio of about 1.9 with RNA samples of both methods, which is close to the optimum value of 2.0. Similar values from 1.5 to 2.0 have also already been measured with saliva samples 26 , 30 . The mean value of the measured A260/230 ratios for estimating the ethanol and salt contamination was about 1.8 for the RNA samples.…”

Section: Discussionsupporting

confidence: 85%

Characterization of non-invasive oropharyngeal samples and nucleic acid isolation for molecular diagnostics

Hose,

Schürmann,

Mennebröcker

et al. 2024

Sci Rep

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Molecular diagnostics is an increasingly important clinical tool, especially in routine sampling. We evaluated two non-invasive methods (oral swabs and mouthwashes) for sampling nucleic acids from the oral/pharyngeal area. We created a workflow from sample collection (n = 59) to RT-qPCR based analysis. The samples were further characterized in terms of their cellular composition as well as the purity, degradation and microbial content of the derived DNA/RNA. We determined the optimal housekeeping genes applicable for these types of samples. The cellular composition indicated that mouthwashes contained more immune cells and bacteria. Even though the protocol was not specifically optimized to extract bacterial RNA it was possible to derive microbial RNA, from both sampling methods. Optimizing the protocol allowed us to generate stable quantities of DNA/RNA. DNA/RNA purity parameters were not significantly different between the two sampling methods. Even though integrity analysis demonstrated a high level of degradation of RNA, corresponding parameters confirmed their sequencing potential. RT-qPCR analysis determined TATA-Box Binding Protein as the most favorable housekeeping gene. In summary, we have developed a robust method suitable for multiple downstream diagnostic techniques. This protocol can be used as a foundation for further research endeavors focusing on developing molecular diagnostics for the oropharyngeal cavity.

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Section: Discussionsupporting

confidence: 85%

Characterization of non-invasive oropharyngeal samples and nucleic acid isolation for molecular diagnostics

Hose,

Schürmann,

Mennebröcker

et al. 2024

Sci Rep

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“…It was suggested that this was due to ribonucleases present in saliva, which could hinder analysis of RNA in the saliva [17]. Recent studies suggested that the method which uses a saliva-specific RNA extraction kit always produces better RNA concentration and yield characteristics as it contained a protecting solution which stabilizes salivary RNA [18]. It therefore was suggested that the low RNA concentration of the saliva samples might be due to the use of a general RNA extraction kit and not a salivaspecific RNA extraction kit.…”

Section: Discussionmentioning

confidence: 99%

Messenger RNA (mRNA)-based age determination using skin-specific markers of saliva epithelial cells

Iroanya

Chioma

Ogunyinka

et al. 2020

Beni-Suef Univ J Basic Appl Sci

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Background Age determination is a vital factor in biological identification in forensics. This study was carried out to determine the expression levels of three target genes (Keratin 9 (KRT9), Loricrin (LOR) and Corneodesmosin (CDSN)) in salivary epithelial cells and how they can be used in age determination using reference gene, β-actin. Thirty young adults participated in the study and were divided into three groups according to their ages (16–20, 21–25, and 26–30). Ribonucleic acid (RNA) extraction, complementary deoxyribonucleic acid (cDNA) synthesis and quantitative polymerase chain reaction (qPCR) were performed. Data analysis was done using IBM SPSS Version 26 and the comparative Ct method (2−∆∆Ct method). Results CDSN was detected in all the sampled age groups. Though the age group 16–20 had the highest (0.4237) expression of CDSN among the three age groups, there was no significant difference (p > 0.05) in the expression of the gene among the three age groups. The LOR gene was lowly expressed across all age groups used in the study. The expression of the gene did not significantly differ (p > 0.05) between the control and 26–30 years age group, but they were however significantly higher (F = 36.47, p ≤ 0.05) than the expression of the gene in both 16–20 and 21–25 years age groups. The KRT9 gene was expressed only in age groups 16–20 and 26–30 and the expression of the gene did not significantly (p > 0.05) differ between these age groups. Though the expression of all the target genes was low, it was observed that the LOR gene expression varied among 21–25 and 26–30 age groups; therefore, more data and further analyses are still required since this experimental approach for age determination using gene expression is still at an emerging stage. Conclusion Although RNA concentration was low and the expression values of the genes were low and could not be used in comparing the expression levels among the three age groups, it can be concluded that the three messenger ribonucleic acid (mRNA) markers CDSN, LOR and KRT9, as well as the ACTB reference mRNA marker analysed via the described qPCR assays, are suitable for identifying epithelial cells in saliva.

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“…The ideal RNA extraction method should be simple, fast, economical, reproducible, with low variability between samples, and able to maintain RNA purity and integrity. Comparative studies from different species [17][18][19][20] and matrices, including saliva, whole blood, and skin [21][22][23][24][25], revealed differences in the yield, quality, and functionality of RNA obtained. However, at present no studies are comparing a substantial number of RNA extraction methods, especially, using the so extensively studied PBMC or human Jurkat T cells.…”

Section: Introductionmentioning

confidence: 99%

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

Ortega-Pinazo

Serrano-Castro

Martínez

et al. 2022

Preprint

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Nucleic acids, RNA among them, are widely used in biomedicine and biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. These data contribute to facilitating researchers or research service platforms in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

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Evaluation of two RNA extraction methods in children's saliva

Cited by 11 publications

References 29 publications

Characterization of non-invasive oropharyngeal samples and nucleic acid isolation for molecular diagnostics

Characterization of non-invasive oropharyngeal samples and nucleic acid isolation for molecular diagnostics

Messenger RNA (mRNA)-based age determination using skin-specific markers of saliva epithelial cells

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

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