RESUMO
Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.
Assuntos
Fasciola hepatica/genética , RNA de Helmintos/análise , RNA Ribossômico 28S/isolamento & purificação , RNA Ribossômico/análise , Animais , Bovinos , Eletroforese em Gel de Ágar , Eletroforese Capilar/métodos , Eletroforese Capilar/normas , Temperatura Alta , Microfluídica , RNA de Helmintos/química , RNA de Helmintos/isolamento & purificação , RNA Ribossômico/química , RNA Ribossômico/isolamento & purificação , RNA Ribossômico 18S/química , RNA Ribossômico 18S/isolamento & purificação , RNA Ribossômico 28S/química , RNA Ribossômico 5,8S/química , RNA Ribossômico 5,8S/isolamento & purificaçãoRESUMO
We provide a simple but very efficient method for RNA preparation from Saccharomyces cerevisiae based on a standard chromosomal DNA isolation protocol. The method yields DNA-free total RNA, including mRNA, rRNA, and tRNA but can easily be adjusted to considerably enrich low molecular weight RNAs, such as tRNAs and the small rRNA species (5S and 5.8S). The procedure was proven and validated by verification of cDNAs belonging to four different genes, two of which encoding polypeptides and two tRNA genes. Besides its simplicity, the method is further advantageous in terms of safety (omitting hazardous phenol) and cost efficiency.
Assuntos
DNA Fúngico/isolamento & purificação , RNA Fúngico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Desoxirribonuclease I/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Ribossômico/isolamento & purificação , RNA Ribossômico 5,8S/isolamento & purificação , RNA Ribossômico 5S/isolamento & purificação , RNA de Transferência/isolamento & purificação , Saccharomyces cerevisiae/metabolismoAssuntos
Pneumopatias Parasitárias/diagnóstico , Tricomoníase/diagnóstico , Trichomonas/genética , Trichomonas/isolamento & purificação , Animais , Primers do DNA , Humanos , Pneumopatias Parasitárias/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/isolamento & purificação , Radiografia Torácica , Tomografia Computadorizada por Raios X , Tricomoníase/diagnóstico por imagemRESUMO
5.8 S rRNA from the gymnosperm Ephedra kokanica Regl. (EMBL Data Library accession No. X15676) has been sequenced. It is 161 nucleotides long and contains three 2'-O-methylated residues--two adenosines and one guanosine. No pseudouridine have been detected. E. kokanica 5.8 S rRNA, as those from other plant species, can form a secondary structure with paired 5'- and 3'-terminal regions. 5.8 S rRNAs of seed plants differ from the moss Mnium reguicum 5.8 S rRNA in that they have longer variable 'GC-rich' hairpins with insertions in the loop region. 5.8 S rRNA of E. kokanica reveals 69 and 82% of homology with that of moss and five angiosperm species, respectively. The posttranscriptional modification pattern of plant 5.8 S rRNAs is not strictly conservative.
