Publicação
Optimization of the protocol: microRNA purification from plasma samples
| Resumo: | Background: MicroRNAs (miRNAs) represent new and potentially relevant diagnostic targets for detection and prognosis of some diseases, like osteoporosis. For that reason, it is critical to have an ideal protocol for its extraction. Here, we are trying to optimize a protocol for miRNA extraction from plasma samples by evaluating different concentrations of glycogen (co-precipitant agent), different initial plasma sample volumes, several elutions and the utilization of DNA low-bind tubes. Results: Comparison of UV-visible spectrophotometry (NanoDrop) results for spiked-in and endogenous miRNA sequences determined extraction efficiencies. Through analyze of PCR results was possible to conclude about the optimal protocol: plasma volume of 200 µL, processed using the QIAGEN miRNeasy Mini Kit, with 5 µg/ml of glycogen, as a co-precipitant, and DNA low-bind tubes. The 3x elution did not show significant advantages. Conclusions: This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized a miRNA extraction protocol. |
|---|---|
| Autores principais: | Gil, Cláudia Raquel Estopa |
| Assunto: | microRNA Glycogen RNA extraction Plasma Diagnosis Mestrado Integrado - 2016 |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Background: MicroRNAs (miRNAs) represent new and potentially relevant diagnostic targets for detection and prognosis of some diseases, like osteoporosis. For that reason, it is critical to have an ideal protocol for its extraction. Here, we are trying to optimize a protocol for miRNA extraction from plasma samples by evaluating different concentrations of glycogen (co-precipitant agent), different initial plasma sample volumes, several elutions and the utilization of DNA low-bind tubes. Results: Comparison of UV-visible spectrophotometry (NanoDrop) results for spiked-in and endogenous miRNA sequences determined extraction efficiencies. Through analyze of PCR results was possible to conclude about the optimal protocol: plasma volume of 200 µL, processed using the QIAGEN miRNeasy Mini Kit, with 5 µg/ml of glycogen, as a co-precipitant, and DNA low-bind tubes. The 3x elution did not show significant advantages. Conclusions: This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized a miRNA extraction protocol. |
|---|