RESUMO
MicroRNAs (miRNAs) are small RNA molecules, which act as post-transcriptional regulators of a gene expression, with important functions within the cell physiology. Whilst many authors have focused on the study of miRNA expression in physiological and pathological processes, various technical variables related to miRNA isolation have simultaneously emerged and the stability of the stored miRNA samples has been questioned. A robust method for RNA isolation is essential for reproducible results and miRNAs instability in the stored samples would make for an alarming situation for most expression studies. Here these issues are discussed and we investigate the stability of miRNAs isolated from clinical samples of B lymphocytes (chronic lymphocytic leukemia) by the most commonly utilized method based on a Trizol/TRI-Reagent solution (RNAs stored at -80 degrees C). To assess the stability of miRNAs, a Real Time-PCR analysis was performed for a panel of 29 miRNAs from a freshly isolated RNA sample and after 14 days storage at -80 degrees C. Furthermore, a Real Time-PCR analysis was repeatedly performed for a stored RNA sample over a period of approximately 10 months. We observed high stability of isolated miRNAs and respective cDNAs. The reproducibility and efficiency of the Trizol/TRI-Reagent isolation method was also tested and compared to the mirVana Isolation kit (Ambion) and RNeasy kit (Qiagen). In conclusion, Trizol/TRI-Reagent based isolation is a robust reproducible method, and obtained miRNA samples do not show any tendency to degradation when properly stored and handled.
Assuntos
MicroRNAs/isolamento & purificação , Estabilidade de RNA , Linfócitos B/química , Guanidinas/química , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , MicroRNAs/química , Fenóis/química , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Soluções , Manejo de EspécimesAssuntos
Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Regulação para Baixo/genética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Masculino , Pessoa de Meia-IdadeRESUMO
Investigations were carried out on the medicines mastiriphin--intramammary syringes, (Mf) in two variants "A" and "B", meant for the treatment of acute mastitis with cows and also mastiriphin depot--intramammary syringes (Mf-depot), as well as two variants with indications for subclinical mastitis cows, containing both medicines in 10 g with 80,000 VI rifampicin, SEC "Pharmachem". Experiments were carried out for determining the stability of the medicines at different pH. The investigations were carried out with 23 lactating and 22 non-lactating cows. Both the degree of retention and the velocity of elimination of rifampicin from the milk and the lactic secretion. It was proved in vitro that rifampicin is stable in a neutral milk milieu (pH 7) in the course of 24 hours, and while in acid or alkaline milieu it was quickly eliminated Mf "A" in the case of intramammary introduction created high bacteriostatic (therapeutic) concentrations in the milk of the lactating animals till the sixth (eighth hour, whereas Mf "B" and the imported analogue rifamasten, provided twice as high levels of antibiotic. Mf-depot "B" introduced in a intramammary way with non-lactating cows was retained in the lactic secretion between 15 and 20 days. The quarantine time limits for using the milk taken from cows, treated in an intramammary way, proved to be 72 hours.