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Background: RNA isolation from bacteria within dentine caries lesions could be difficult due to reduced amount of collectable biomass and high mRNA instability. Attempting to overcome this challenge we describe one protocol developed to extract and purify total RNA from dentine lesions. Objective: customize a bacterial RNA extraction and purification method from human carious dentine. Methods: quantity and purity of extracted RNA were measured with a microvolume UV-VIS spectrophotometer, RNA integrity was assessed by standard denaturing agarose gel electrophoresis and images were captured under ultraviolet light with camera and analyzed. DNase treatment removed genomic DNA and an additional step of purification was carried out in silica spin column. Results: final yield (ng/µl) was 67.01 ± 22.33, absorbance ratio A260/A280 = 2.0 ± 0.07 and RNA integrity were obtained. The purified samples were reversely transcribed and the expression of atpD and fabM gene from Streptococcus mutans analyzed by quantitative real-time PCR. Conclusion: the extraction methodology developed produced high-quality RNA from dentine microbiota for transcriptional analysis.

Introdução: o isolamento de RNA de bactérias dentro de lesões de dentina cariada pode ser difícil devido à quantidade reduzida de biomassa e alta instabilidade de mRNA. Na tentativa de superar esse desafio, descrevemos um protocolo desenvolvido para extrair e purificar o RNA total das lesões dentinárias. Objetivo: personalizar um método de extração e purificação de RNA bacteriano a partir da dentina cariada humana. Métodos: a quantidade e a pureza do RNA extraído foram medidas com um espectrofotômetro UV-VIS de microvolume, a integridade do RNA foi avaliada por eletroforese em gel de agarose desnaturante padrão e as imagens foram capturadas sob luz ultravioleta e analisadas. O tratamento com DNase removeu o DNA genômico e uma etapa adicional de purificação foi realizada em coluna de spin de sílica. Resultados: o rendimento final (ng / µl) foi de 67,01 ± 22,33, a razão de absorbância A260 / A280 = 2,0 ± 0,07 e a integridade do RNA foram obtidas. As amostras purificadas foram transcritas reversamente e a expressão do gene atpD e fabM de Streptococcus mutans analisadas por PCR quantitativo em tempo real (RT-qPCR). Conclusão: a metodologia de extração desenvolvida produziu RNA de alta qualidade da microbiota dentinária para análises transcricionais.

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Several studies of antimicrobial photodynamic therapy (aPDT) have been performed to verify the efficiency of this treatment against caries-related microorganisms. Thus, the aim of this study is to describe the characteristics of aPDT and to review the literature regarding its effects on cariogenic microorganisms organized in biofilms and/or caries lesions. The literature was searched for reviews and original papers about aPDT and its outcomes against Streptococcus mutans as well as other caries-associated microorganisms or caries lesions. Moreover, research on photosensitizers and light sources are also reviewed. The publications were selected using PubMed, Web of Science, and manual search of references cited in key papers. The descriptors used were "dental caries" and "photodynamic therapy". The relative efficacy of aPDT to reduce the population of cariogenic bacteria in in vitro biofilms is demonstrated by large number of laboratory studies. Preclinical (in situ models) and clinical studies show a less pronounced bacterial reduction for aPDT than for in vitro models, especially in dentin carious lesions, since the bacteria in dentin caries may be less susceptible to this therapy due to the limited photosensitizer penetration as well as reduced diffusion of light along dentin structures. Although aPDT may be an efficient and less invasive complementary approach to disinfect deep caries lesions, there is insufficient scientific evidence of its efficacy to warrant a clinical recommendation for its use.

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