cdna synthesis for real time pcr

... assessment For real- time PCR the cDNA was amplified using the MyiQ Real- Time PCR Detection System (Bio-Rad, Hercules, CA, USA) The reverse transcription product was subjected to real- time PCR in ... the ThermoScript™ RT -PCR system (Invitrogen) for first-strand cDNA synthesis in 50 μg reaction volume For semiquantitative PCR, each reverse transcription sample was assessed for glyceraldehyde ... glyceraldehyde 3-phosphate dehydrogenase cDNA The cDNA was amplified by PCR using 24 to 32 cycles of 95°C for 30 seconds, 55°C to 60°C for 30 seconds, and 72°C for 30 seconds in the presence of Taq...

Ngày tải lên: 09/08/2014, 10:23

... extended our knowledge of NO-mediated Fe mobilization For the first time, we demonstrate using a cellular system that NO intercepts Fe before being incorporated into ferritin in a similar manner ... centrifuged at 21 300 g for 30 at °C and the cytosol removed and assessed for radioactivity using the c-counter described above The cytosolic samples were then incubated for h at 37 °C with DFO ... SK-N-MC neuroepithelioma cells were labelled for h at 37 °C with 59Fe-transferrin (0.75 lM) and washed four times on ice The cells were then reincubated for h at 37 °C with control media, GSNO (0.5...

Ngày tải lên: 08/03/2014, 22:20

... lectin domains of these isoforms are responsible for the unique GalNAcglycopeptide specificities [41] We therefore hypothesized that different subsets of pp-GalNAc-T isoforms are designated to generate ... Research Association for Biotechnology, the Program for the Promotion of Basic Research Activities for Innovative Biosciences, and the Danish Cancer Society We thank C Hiraiwa for her assistance ... has been proposed recently that some GalNAc-T isoforms function as follow-up enzymes in that they are directed by the initial action of other isoforms [21,23,28,35,40] This latter mechanism is...

Ngày tải lên: 17/03/2014, 10:20

... before PMA treatment Dose-dependent O2–-scavenging effects of the compound were found by eliminating MCLA-dependent CL Arrows indicate the time at which MCLA or PMA was added O2– was recorded for ... 3.7% formaldehyde in NaCl ⁄ Pi (v ⁄ v) was added carefully to each dish for 10 at room temperature (25 °C) to fix the attached cells The cells were then washed gently with NaCl ⁄ Pi three times ... mL of 0.1% Triton X-100 in NaCl ⁄ Pi in each dish for 10 After permeabilization, cells were washed three times with NaCl ⁄ Pi, and incubated for 30 in darkness at °C with a primary antibody (antibody...

Ngày tải lên: 23/03/2014, 07:20

... black-adapted Xenopus were preincubated overnight, pulsed for h with [35S]Met/Cys and chased for the indicated time periods The incubations were performed either in the absence or presence of 10 lgÆmL)1 ... cleavage fragments NILs from black-adapted Xenopus were pulsed for h with [35S]Met/Cys and subsequently chased for the indicated time periods in the presence of BFA Ac45 products were immunoprecipitated ... C-terminal fragment of Ac45 NILs from black-adapted Xenopus were pulsed for h with [35S]Met/Cys and subsequently chased for the indicated time periods in the presence of NDGA or BFA Ac45 products were...

Ngày tải lên: 31/03/2014, 09:20

... parallel conformation based on best fit to the experimental data were selected Each of these conformations was used as input into the Rosetta Dock server as the starting conformations for refinement ... with PBS and fixed in 3.7% formaldehyde at room temperature for 10 minutes, and washed and permeabilized with 0.5% Triton X-100 for an additional 10 minutes After washing times with PBS, cells were ... play a pivotal role in mediating higher order Vpr-Vpr interactions We therefore built models for higher order multimeric forms, and a hexamer model (Fig 3C and Fig 3D) exhibited an interface where...

Ngày tải lên: 12/08/2014, 04:20

... coding for wild type or mutant forms of hA3G The total amount of plasmid DNA used for transfection was kept constant in controls by replacing plasmid coding for hA3G with the empty vector, pcDNA3.1 ... into the HMM form This suggests that the RA LMM hA3G, instead of the LMM form, acts as the precursor of the HMM hA3G complex All this reflects a dynamic course among LMM, RA LMM and HMM forms of ... means of results of experiments performed at least three times, and the error bars represent standard deviations in Figure The amounts of hA3G in these three forms were determined by Western blot...

Ngày tải lên: 13/08/2014, 01:20

... ATG GTG-3’; reverse, 5’-CTC ACA TTT GCT TGG TTG TC-3’) Real- time PCR was performed with the iQ SYBER Green kit (BioRad) using the CFX96 real- time system as previously described [6] Statistical analysis ... of IFIT-1, an IFN stimulated gene (ISG), in WT HIV-1 NL(AD8) or ΔNef-infected iDCs using real- time RT -PCR Compared with mock infection, WT and ΔNef HIV-1 infections in iDCs resulted in a 5-fold ... mRNA in iDCs mock infected or infected with WT NL(AD8) or NL(AD8)ΔNef were quantified using real- time RT -PCR The IFIT-1 expression levels were normalized by the relative GAPDH expression levels...

Ngày tải lên: 13/08/2014, 01:20

... carried out at 37 °C for 60 and followed by denaturation at 95 °C Two microliters of the ®rst strand synthesis product (0.1 lg) was used as template to amplify each cDNA PCR was performed with 250 ... P450 aromatase cDNA was ampli®ed at 94 °C for for 30 cycles as described previously [30], which consisted of a denaturating step (at 94 °C for min), an annealing step (at 60 °C for 30 s) and an ... extension step (at 72 °C for min) then a ®nal elongation step (at 72 °C for 10 min) in ROBOCYCLERÒ Gradient 40 (Stratagene) To check for contaminating genomic DNA, a RT -PCR was performed on RNA without...

Ngày tải lên: 21/02/2014, 03:20

... pCRLPs for 48 h and the levels of mRNA for nine genes believed to play a role in foam cell formation was measured by RT -PCR The results are shown in Table The relative abundance of transcripts for ... chylomicron remnant-like particles (CRLPs) or pCRLPs (30 lgÆmL21 cholesterol) for h (A, CRLPs; B, pCRLPs), h (C, CRLPs; D, pCRLPs) or 24 h (E, CRLPs; F, pCRLPs), and viewed by confocal microscopy ... containing probucol (pCRLPs) on mRNA levels for genes involved in foam cell formation THP-1 macrophages were incubated with or without CRLPs or pCRLPs (30 lgÆmL)1 of cholesterol) for 48 h, and the...

Ngày tải lên: 16/03/2014, 16:20

... 19221.7 – Tyr 19249.7 – formyl + Tyr 19238.4 – DOPA 19254.0 – DOPA 19269.3 – DOPA 19281.9 – formyl + DOPA 19297.9 – formyl + DOPA 19320.0 – formyl + DOPA + Na 19338.2 – formyl + DOPA + K Fig Positive-ion ... protein synthesis turns out to provide a fast route for the qualitative detection of the formation of protein aggregates, as insoluble proteins form visible precipitates already during protein synthesis ... following their in vitro synthesis Results Protein synthesis in the presence of DOPA The effect of substitution of DOPA for tyrosine was investigated in a preparative in vitro protein synthesis FEBS Journal...

Ngày tải lên: 16/03/2014, 22:20

... 70 kDa form of the enzyme (using the antibody against the hinge region, data not shown) The released forms of MMP-9 from the heterodimer are active Fig Time dependent release of low Mr forms of ... low Mr forms from the complex, while for most preparations release of low Mr forms was obtained after h incubation It is known that CS chains can bind various proteins [40] The variation in time ... 74/76 kDa, 80/85 kDa and 100 kDa forms of MMP-9 To determine which of these forms were active in solution, proMMP-9/CSPG complex was incubated with CaCl2 at various time intervals and then mixed...

Ngày tải lên: 17/03/2014, 10:20

... GTP, 40% glycerol) for 30 min, then centrifuged at 100 000 g for 10 at 27 °C The pellet and supernatant fractions were processed to measure radioactivity bound to protein, and for immunoblot using ... alters the ability of the molecule to form microtubules For this purpose, we studied the behavior of nitrotyrosinated tubulin (and of tyrosinated tubulin for comparison) during reconstitution ... Carlos E Argarana and Mario Guido for critical ˜ reading of the manuscript, Mrs S N Deza and Mrs M G Schachner for technical assistance and Dr Stephen Anderson for English editing of the manuscript...

Ngày tải lên: 17/03/2014, 10:20

... interaction with lipoproteins is stronger for LolB than for LolA [6] Moreover, the extra C-terminal loop characteristic of LolA has been found to be important for the prevention of the re-incorporation ... Lpp, Pal, BamD (formerly YfiO) and LptE (formerly RlpB), were correctly localized in the outer membrane, whereas the inner membrane-specific AcrA remained in the inner membrane Therefore, lipoproteins ... (%) 80 60 40 20 0 10 20 PG/PE 60 CL 40 PG 20 30 CL/PE 80 10 20 Time (min) Time (min) Phosphatidylethanolamine (PE) is important for LolB function Escherichia coli membranes contain PE, phosphatidylglycerol...

Ngày tải lên: 23/03/2014, 05:22

... current study reflect the events that occur in vivo for the formation of the insoluble fibers of the PL Our results also provide evidence, for the first time, that serum ZP1 can interact with both ZP3 ... this interaction appears to be involved in the formation of the insoluble fibers of the PL [18,19] In the present study, we showed, for the first time, that exogenous ZP1 purified from the serum ... polyacrylamide for resolving and stacking gels, respectively For western blotting, proteins separated on SDS-PAGE were transferred to a PVDF membrane (Immobilon-P; Millipore Bedford, MA, USA)...

Ngày tải lên: 23/03/2014, 07:20

... vector that may be useful for clinical gene therapy applications, especially for applications involving gene delivery into low-CAR expressing cells Page of 11 (page number not for citation purposes) ... and is too large for direct modification using conventional cloning techniques To achieve our goal, we therefore established a bacteria-based homologous recombination system for Ad5 fiber modification ... cells were cultured for two days in the humidified 37°C, 5% CO2 incubator, and GFP microscopy was performed to examine the transgene expression Page of 11 (page number not for citation purposes)...

Ngày tải lên: 18/06/2014, 18:20

... sulfate (SDS) and extracted three times with phenol/chloroform The RNA was precipitated with Polyethylene glycol (PEG), reextracted three times with penol/chloroform, and precipitated with 2.5 ... + + + + + + + + - + + + + + + - formed as described Generated viruses were isolated using plaque assays on L-ST3.S2 cell monolayers, and replaqued three times for purity The dsRNA genome segments ... model for genome assortment and replication Using the minimal 5' and 3' sequences we have now identified for the s2 ssRNA, it is possible to use this marker res- Page of 11 (page number not for...

Ngày tải lên: 19/06/2014, 08:20

... vector that may be useful for clinical gene therapy applications, especially for applications involving gene delivery into low-CAR expressing cells Page of 11 (page number not for citation purposes) ... and is too large for direct modification using conventional cloning techniques To achieve our goal, we therefore established a bacteria-based homologous recombination system for Ad5 fiber modification ... cells were cultured for two days in the humidified 37°C, 5% CO2 incubator, and GFP microscopy was performed to examine the transgene expression Page of 11 (page number not for citation purposes)...

Ngày tải lên: 20/06/2014, 01:20

... sodium chlorate was performed for 24 hours before CXCL12α binding Removal of surface HSPGs in cultured Available online http://arthritis-research.com/content/8/2/R43 Figure For immunolabelling of ... ml LT-α1/β2 (R&D Systems, Inc., Abingdon, Oxon, UK) for 16 hours before CXCL12α binding CXCL12 expression by ECs was studied by RT -PCR on cDNA synthesized from µg of total RNA with the use of ... cluster located in the first Page of (page number not for citation purposes) sheet of the CXCL12 β strand, which forms the canonical binding motif for HSPG Materials and methods Biotinylated CXCL12α...

Ngày tải lên: 09/08/2014, 07:20