(B) Western blotting analyses of caspase 3, 7, 8, 9, Bcl-2 and Bax in melanoma tissue. The mice were sacrificed and analyzed 1 or 7 days after BNCT treatment. On the fourteenth day after B16F10 melanoma implantation, the BNCT group received BPA and was irradiated with thermal site neutrons. (A) Tumor volume of mice bearing B16F10 melanoma during twenty-one days. qRT-PCR quantification of expression of the target gene in tumorApoptosis in Melanoma Cells after BNCTFigure 6. To evaluate the amplification efficiency of each target and housekeeping gene (HPRT), standard curves were constructed from a reference sample of RNA using duplicate serial dilutions at five Hypericin different RNA concentrations (0.8, 4, 20, 100, and 500 ng/mL). Amplified fragment sizes were 122, 118 and 145 bp for Caspase 3, Caspase 8 and HPRT, respectively. The nucleotide sequences for the primers were: Caspase 3 (NM_009810.2) forward 59- TGA CTG GAA AGC CGA AAC TC 39 and reverse 59- AGC CTC CAC CGG TAT CTT CTV39 Caspase 8 (NM_009812.2) forward 59- CCG AGC TGG ACT TGT GACC -39 and reverse 59- CTG CCC AGT TCT TCA GCA AT-39 and HPRT (NM_013556.2) forward 59 CTT CCT CCT CAG ACC GCT TT -39 and reverse 59- TTT VCCA AAT CCT CGG CAT AA – 39. Primers were designed to have similar GC content and melting temperatures using the Primer3_cgi v 0.2 program. Gene-specific primer pairs were located on two adjacent exons to achieve a high level of specificity and to avoid detection of genomic DNA. Specificity of amplicons was also ensuredby agarose gel electrophoresis to visualize a unique product fragment of appropriate size. Fluorescence changes were monitored after each cycle, and melting curve analysis was performed at the end of cycles to verify PCR product identity (72uC ramping to 99uC at 0.2uC/second with continuous fluorescence readings). Reactions were carried out under the following cycling conditions: 50uC for 30 min, 95uC for 15 min, and 40 cycles of 95uC for 20 s, followed by 56uC for 30 s and 72uC for 30 s before a final primer sequence extension incubation at 72uC for 5 min. Negative samples were run for each qRT-PCR assay consisting of no RNA in the reverse transcriptase reaction and no cDNA in the PCR. Reaction mixture contained 50 ng of total RNA from each sample, 1.0 mM 25033180 of each primer, 12.5 mL of 2X SYBRH Green Reaction Mix and 0.5 mL of SuperScriptTM III RT/ PlatinumH Taq Mix to a final volume of 25 mL. The reactions were prepared according to standard protocols. Merase Chain Reaction (qRTPCR) AnalysisReactions were conducted on a Rotor-GeneTM 6000 (Corbett Research, Sydney, NSW, Australia) using a SuperScriptTM III PlatinumH SYBRH Green One-Step qRT-PCR kit (Invitrogen Corporation). By directly controlling all phases of production, we optimize quality control, minimize overhead costs and ensure the most rapid product development cycles.By Src inhibitor- srcinhibitor August 21, 2017 Our positioning in the biotech manufacturing sector has enabled us to establish a reputation for continuous and rapid in-house innovation and product evolution. Our range of instrument systems has grown to include the next generation Rotor-Gene6000 series real-time analyzers CAS-1200 Precision Liquid Handling Workstations, the X-tractor Gene fully-automated 96-well nucleic acid extraction system, the Palm-Cycler gradient thermal cycler with a palmtop computer interface, and the Gel-Scan 3000 automated DNA Sequencer and Fragment Analyzer. The renowned Rotor-Gene software now supports the widest application set. It also features high-speed data capture and the broadest optical range of any real-time instrument and now High Resolution Melt (HRM) capability. The Rotor-Gene is the most thermally and optically precise real-time analyzer available. We are perhaps most recognized for developing the world's first rotary real-time DNA amplification system-the Rotor-Gene. Corbett Robotics Inc (San Francisco, USA).Corbett Robotics Pty Ltd (Brisbane, Australia).Corbett Research UK Ltd (Cambridge, United Kingdom).Corbett Research Pty Ltd (Sydney, Australia). ![]() Corbett Life Science comprises four operating units:.Since 1988, we have earned a reputation for innovative, versatile, robust, and easy-to-use products that generate strong customer loyalty. LC-MS innovation: Improve analytical accuracyĬorbett Life Science manages a group of biotechnology companies that design, manufacture and internationally distribute instrumentation systems for the life sciences. Which microbioreactor modules are best for you Which solid support is right for your oligo synthesis? Cell & Gene Therapy Accelerating ScienceĬell & Gene Therapy Accelerating Science Feature.SLAS2024 International Conference and Exhibition.
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