Project description:we designed a CRISPR-based chromosome-doubling technique to construct an artificial diploid Escherichia coli cell. The stable diploid E. coli was confirmed by quantitative PCR and third-generation genome sequencing.
Project description:We checked the effects of mutations on expression levels by using systematically mutated probes. We used Agilent custom microarray whose probe length is 60bp. To check artificial mutation effect, we designed 56 patterns of mutations, 24 patterns of insertions and deletions. On the microarray, we used 7,987 probes from Agilent human gene probes. In these experiments, we checked the difference of expression levels about at least 73 probes for each mutation type. We performed microarray experiments with an Agilent 4x44k custom microarray. We measured gene expressions using a qPCR Human reference total RNA sample provided by CloneTech. Gene expression profiles were normalized with quintile normalization according to the Agilent protocol except that only non-mutated 219 probes were used.
Project description:We checked the effects of mutations on expression levels by using systematically mutated probes. We used Agilent custom microarray whose probe length is 60bp. To check artificial mutation effect, we designed 56 patterns of mutations, 24 patterns of insertions and deletions. On the microarray, we used 7,987 probes from Agilent human gene probes. In these experiments, we checked the difference of expression levels about at least 73 probes for each mutation type. We performed microarray experiments with an Agilent custom microarray. We measured gene expressions using a qPCR Human reference total RNA sample provided by CloneTech. Gene expression profiles were normalized with quintile normalization according to the Agilent protocol except that only non-mutated 219 probes were used.
Project description:Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead–based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile (ACN), and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈ 1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, rat bile, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. Notwithstanding, we must highlight the performance of this method in the preparation of a challenging biological fluid as bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction of digestion of bile proteins, our method allowed to identify exclusively 9.91% (x proteins), thus contributing to improving the coverage of the bile proteome.
Project description:Purpose: Next-generation sequencing (NGS) on targeted locus in Sindbis genome to determine frequency changes of artificial microRNAs expressed by viruses after passaging in cancer and normal cells Methods: RNA was harvested in Trizol 488 (Thermo Fisher). RNA was extracted using the manufacturer’s protocol and quantified by nanodrop. Sequencing was done by SeqMatic on a MiSeq v3 platform generating 75bp reads. Adapters were trimmed using Trimmomatic and adapter-free reads represent artificial microRNAs encoded by Sindbis virus in a sample. Results: We have identified changes in artificial microRNA frequency after passaging virus pool in cancer and normal cells and have identified microRNAs increasing viral fitness in cancer cells. Conclusions: Our study represents artificial microRNAs which target pathways that can aid oncolytic viral replication in cancer cells.
Project description:We checked the effects of mutations on expression levels by using systematically mutated probes. We used Agilent custom microarray whose probe length is 60bp. To check artificial mutation effect, we designed 56 patterns of mutations, 24 patterns of insertions and deletions. On the microarray, we used 7,987 probes from Agilent human gene probes. In these experiments, we checked the difference of expression levels about at least 73 probes for each mutation type. We performed microarray experiments with an Agilent 4x44k custom microarray. We measured gene expressions using a qPCR Human reference total RNA sample provided by CloneTech. Gene expression profiles were normalized with quintile normalization according to the Agilent protocol except that only non-mutated 219 probes were used. 6 replicate samples
Project description:We checked the effects of mutations on expression levels by using systematically mutated probes. We used Agilent custom microarray whose probe length is 60bp. To check artificial mutation effect, we designed 56 patterns of mutations, 24 patterns of insertions and deletions. On the microarray, we used 7,987 probes from Agilent human gene probes. In these experiments, we checked the difference of expression levels about at least 73 probes for each mutation type. We performed microarray experiments with an Agilent custom microarray. We measured gene expressions using a qPCR Human reference total RNA sample provided by CloneTech. Gene expression profiles were normalized with quintile normalization according to the Agilent protocol except that only non-mutated 219 probes were used. 6 replicate samples