RESUMEN
MicroRNAs are a class of small noncoding RNA which play important regulatory roles in a variety of cancers. MiRNA-specific expression profiles have been reported for several pathological conditions. In this study, we combined large scale parallel Solexa sequencing to identify 11 up-regulated miRNAs and 19 down-regulated miRNAs with computational techniques in the sera of ovarian cancer patients while using healthy serum as the control. Among the above, four miRNAs (miR-22, miR-93, miR-106b, miR-451) were validated by quantitative RT-PCR and found to be significantly aberrantly expressed in the serum of ovarian cancer patients (P<0.05). There were no significant differences between samples from cancer stage I/II and III/IV. However, the levels of miR-106b (p=0.003) and miR-451 (p=0.007) were significantly different in those patients under and over 51 yearsof age. MiR-451 and miR-93 were also specific when analyzed with reference to different levels of CA125. This study shows that Solexa sequencing provides a promising method for cancer-related miRNA profiling, and selectively expressed miRNAs could be used as potential serum-based biomarkers for ovarian cancer diagnosis.
Asunto(s)
MicroARNs/sangre , Neoplasias Ováricas/sangre , Secuencia de Bases , Biomarcadores de Tumor/sangre , Antígeno Ca-125/sangre , Femenino , Humanos , Masculino , Proteínas de la Membrana/sangre , MicroARNs/biosíntesis , Persona de Mediana Edad , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/genética , Análisis de Secuencia de ADNRESUMEN
Pools of short synthetic oligonucleotides (oligos) are required in the multiplex and parallel DNA construction. Microarray technology provides a fast and economical mean for massive parallel synthesis of oligos. The method of oligo synthesis with the programmable microfluidic PicoArray could simultaneously synthesize the designed oligos for multiple riboswitch genes. The synthetic oligos were recovered and purified as a pool of oligo mixture (OligoMix). Three temperature steps were employed to denature, anneal and extend the designed OligoMix until, after multiple rounds of thermocycling, the riboswitches with the desired length are obtained. The OligoMix was amplified using this PCR-based technique and the flanking adapter segments were cleaved for following assembly. Based on these oligos derived from 197 riboswitch sequences, the method of simultaneous assembling multiplex riboswitches (SAMRs) showed high fidelity by sequence identification. The resultant error rate was determined to be 2.78 per thousand. With the templates from SAMRs, in vitro transcription was applied to produce milligram amounts of biologically active riboswitches. With the verification of biological activity based on the high specificity of recognizing small-molecule metabolites as well as the DNA sequence redivivus by RT-PCR, the assembled riboswitches can be used for further gene operation and biological application.
Asunto(s)
Clonación Molecular/métodos , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Aptámeros de Nucleótidos/líquido cefalorraquídeo , Aptámeros de Nucleótidos/metabolismo , Electroforesis en Gel de Agar , Ligandos , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A method for 2-SH2 protein domain study was described as per the order of expression, purification and structural detection. The 2-SH2 protein of Homo sapiens SHP-2 was successfully expressed and purified. It could specifically bind to anti-SHP-2/SHPTP-2 antibody according to the MS and Western blot analysis. The NMR spectrum result reveals that the protein exists in a well-ordered structure. This can provide foundations to find out the reaction mechanism of the D phosphorylated-EPIYA motif accessible to 2-SH2, support the research and development of the novel detection chip as well as target inhibition medicine for the future clinical applications.
Asunto(s)
Estructura Terciaria de Proteína/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/aislamiento & purificación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN/genética , Escherichia coli , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Análisis de Secuencia de ADN , Difracción de Rayos XRESUMEN
Uptake HTO from seawater by five kinds seashell and formative dynamics of bound tritium in organ of seashell are studied by using the isotope-tracer techniques in order to get a better understanding of the marine environmental behavior of HTO. The results show that the tritium in seashell exists in free water tritium and bound tritium form. The free water tritium is main form; its content observed was more than 97.4% of total tritium. The bound tritium content was very lower, only being 0.4% - 2.6% in total tritium. The speed of HTO untaken by seashell is very rapid, specific activity of HTO in three kinds seashell had reached their maximum only 2 h after treatment. The bound tritium in seashell increases slowly with time. The results of analysis of concentration factor (CF) values indicate that HTO would not concentrate in the seashell.
