ผลต่างระหว่างรุ่นของ "หน้าหลัก"

จาก wiki.surinsanghasociety
ไปยังการนำทาง ไปยังการค้นหา
ล
ล
แถว 1: แถว 1:
−
Om control and individuals were cultured for an added 18 h simultaneously in normoxia conditions (20  O2) at the same time inside a 1  O2 hypoxic chamber.QPCR validation12.5 ng of total RNA from 11 ALS cases, six PLS instances and 10 controls (Table S1) was reverse transcribed employing Superscript III and oligodT primers as per the makers protocol (Invitrogen, Life Technologies, Paisley, UK) and employed together with 1Brilliant II SYBR Green PCR Master Mix (Stratagene, Agilent Technologies, Cheadle, UK) and optimized concentrations of forward and reverse primers in each assay (Table S2). The assays have been run on an MX3000P RealTime PCR method (Stratagene) with acceptable RT negative reactions without having Superscript III. For every single gene of interest, the fluorescent signal intensity was analysed utilizing the MxPro software (Stratagene) and its gene expression value normalized to housekeeping gene betaactin (ACTB) employing the 2Ct approach (ABI PRISM 7700 Sequence Detection Method protocol, Applied Biosystems, Warrington, UK). The ACTB gene was chosen as a appropriate housekeeping gene on account of its steady expression across all patient and manage fibroblasts around the microarray chips. Expression levels for every single gene are expressed relative to their expression levels in handle fibroblasts. Statistical significance of expression levelsNAN 2015; 41: 201RNA extraction, amplification, fragmentation and labellingRNA was isolated from 6 SALS, 6 PLS and 6 handle fibroblast cultures applying the RNeasy Mini Kit (Qiagen, Manchester, UK) as per the manufacturer's protocol. RNA was linearly amplified and biotin labelled using the OneCycle Affymetrix Labelling Kit (Affymetrix, High Wycombe, UK) as per the manufacturer's protocol. Biotinlabelled cRNA was fragmented to 3500 bp fragments by heating to 95  for 35 min with a fragmentation buffer (Affymetrix) and subsequently hybridized overnight at 42  to Human U133 Plus two.0 GeneChips as per the Eukaryotic Target Hybridisation protocol (Affymetrix). GeneChips were stained and washed inside a GeneChip Fluidics Station 400 (Affymetrix). The fluorescence intensity of hybridized transcripts was determined together with the highresolution laser of the GeneChip 3000 Scanner (MegAllele system, Affymetrix).2014 The Authors. Neuropathology and Applied Neurobiology published by John Wiley Sons Ltd on behalf of British Neuropathological SocietyR. Raman et al.involving disease and controls was assessed on applying an unpaired Student's ttest employing [http://sc.bodaxing.com/comment/html/?285281.html SFigure 1. Systematic evaluation of RISCIP from HCMV infected fibroblast cells. (A] GraphPad Prism six (GraphPad Computer software Inc., California, USA).Metabolic measurements working with Seahorse assaysFibroblasts from 6 SALS patients and 9 controls (Table S1) had been plated at 50 000 cellswell in a 24 nicely Seahorse cell culture plate (Seahorse Bioscience, Copenhagen, Denmark) in 250 l minimal media (PAA) supplemented with 10  FCS gold (PAA), two mM glutamine (Lonza, Basel, Switzerland), 50 gml uridine (Sigma), 100vitamins (Lonza five ml in 500 ml), 100amino acids (Lonza five ml in 500 ml), sodium pyruvate (Lonza five ml in 500 ml) and penicillinstreptomycin (Lonza five ml in 500 ml 5000 Uml). The cells were incubated at 37 5  CO2 overnight. The following day the media was removed plus the cells had been washed with 1000 l XF Assay Media pH 7.four (Seahorse Bioscience) supplemented with 1 mM sodium pyruvate, two mM glutamine and 1mgml Dglucose (Sigma). Cells had been incubated at 37  inside a non CO2 incubator for 1 h within a total volume of 675 l of XF Assay media.
+
Mgml. 5.0 M FCCP (Sigma), 0.five M final concentration and five.0 M rotenone (Sigma), final concentration 0.5 M. All have been made up in assay media as described above. The microplate was calibrated within a Seahorse XF24 analyser prior to addition from the cell culture plate. 3 basal oxygen consumption rate (OCR) and extracellular acidification price (ECAR) measurements had been recorded prior to addition on the mitochondrial inhibitors. The cells have been measured three occasions for 3 min each and every. Cell quantity was normalized by addition of two.5 M calcein (Invitrogen) incubated using the cells for 60 min and fluorescence measured on a Fluorostar Omega plate reader (BMG Labtech, Ortenberg, Germany) at Ex485nmEm520nm.miRNA specific stem loop primers (Applied Biosystems). Following subsequent preamplification, the expression of miRNA was measured making use of QPCR with TLDA cards (Human A v3.0). The card information have been analysed on an ABI 7900HT QPCR program using Sequence Detection Method (SDS) software program v2.3 based on the manufacturer's encouraged situations. Manual inspection of amplification plots and preliminary data evaluation have been performed applying SDS RQ manager v1.2 and Data Assist software v2.0 respectively (Applied Biosystems). Relative expression of mature miRNAs was calculated working with the competitive CT 2Ct process, with stably expressed miRNAs across all samples, (as identified applying NormFinder algorithm [19], acting as controls to normalize the information.Immunoblotting for hypoxic response in fibroblastsTotal protein was extracted by washing cells twice in PBS and scraping into 100 l cell lysis RIPA buffer (150 mM NaCl, 1  IGEPALCA630, 0.5  sodium deoxycholate, 0.1  SDS, 50 mM Tris pH eight.0, plus protease inhibitor cocktail) (Sigma) at 4 . Protein concentration was determined by the colorimetric Bradford assay utilizing Coomassie Brilliant Blue dye (BioRad, Hemel Hempstead, UK). 80 g of total protein in addition to prestained protein ladder (Bioline, London, UK) was separated on 10  sodium dodecyl sulphate polyacrylamide gel (SDSPAGE). Protein samples were transferred overnight onto polyvinylidene difluoride (PVDF) membrane (Millipore, Watford, UK) and blocked in PBST and 5  (wv) dried skimmed milk. The blots have been then probed in PBST plus milk with mouse polyclonal anti HIF1 (R D Biosciences, Abingdon, UK) diluted 1:500, rabbit polyclonal antiPHD2 (Abcam, Cambridge, UK) diluted 1:1000 and rabbit polyclonal antitubulin (Abcam) diluted 1:5000. To detect HIF1, the blot was incubated for four h at space temperature; to detect PHD2 and tubulin incubation was for 1 h at space temperature. Subsequently, every single blot was probed for 1 h at space temperature with peroxidase conjugated goat antimouse secondary antibody diluted 1:6000 (for HIF1) or goat antirabbit secondary antibody diluted 1:4000 (for PHD2 and tubulin). Antibody bound to protein was detected by enhanced chemiluminescence (ECL) kit (Amersham, GE Healthcare Life Sciences, Tiny Chalfont, UK) as per the manufacturer's protocol. Protein quantification was carried out utilizing GBoxHR Gel Doc technique (Syngene, Cambridge, UK).NAN 2015; 41: 201MicroRNA profiling of fibroblastsTotal RNA was extracted from 6 SALS and six control T75 fibroblast culture flasks making use of the mirVana [http://sczmsp.com/comment/html/?283978.html Ations in about 500 HGSC samples [2]. By means of computational analysis of TCGA data] isolation kit (Applied Biosystems) based on the manufacture's protocol. TaqManLow Density Arrays (TLDAs) had been utilized to measure the expression amount of 377 microRNAs (miRNAs) (Applied Biosystems).

รุ่นแก้ไขเมื่อ 07:49, 22 เมษายน 2564

Mgml. 5.0 M FCCP (Sigma), 0.five M final concentration and five.0 M rotenone (Sigma), final concentration 0.5 M. All have been made up in assay media as described above. The microplate was calibrated within a Seahorse XF24 analyser prior to addition from the cell culture plate. 3 basal oxygen consumption rate (OCR) and extracellular acidification price (ECAR) measurements had been recorded prior to addition on the mitochondrial inhibitors. The cells have been measured three occasions for 3 min each and every. Cell quantity was normalized by addition of two.5 M calcein (Invitrogen) incubated using the cells for 60 min and fluorescence measured on a Fluorostar Omega plate reader (BMG Labtech, Ortenberg, Germany) at Ex485nmEm520nm.miRNA specific stem loop primers (Applied Biosystems). Following subsequent preamplification, the expression of miRNA was measured making use of QPCR with TLDA cards (Human A v3.0). The card information have been analysed on an ABI 7900HT QPCR program using Sequence Detection Method (SDS) software program v2.3 based on the manufacturer's encouraged situations. Manual inspection of amplification plots and preliminary data evaluation have been performed applying SDS RQ manager v1.2 and Data Assist software v2.0 respectively (Applied Biosystems). Relative expression of mature miRNAs was calculated working with the competitive CT 2Ct process, with stably expressed miRNAs across all samples, (as identified applying NormFinder algorithm [19], acting as controls to normalize the information.Immunoblotting for hypoxic response in fibroblastsTotal protein was extracted by washing cells twice in PBS and scraping into 100 l cell lysis RIPA buffer (150 mM NaCl, 1 IGEPALCA630, 0.5 sodium deoxycholate, 0.1 SDS, 50 mM Tris pH eight.0, plus protease inhibitor cocktail) (Sigma) at 4 . Protein concentration was determined by the colorimetric Bradford assay utilizing Coomassie Brilliant Blue dye (BioRad, Hemel Hempstead, UK). 80 g of total protein in addition to prestained protein ladder (Bioline, London, UK) was separated on 10 sodium dodecyl sulphate polyacrylamide gel (SDSPAGE). Protein samples were transferred overnight onto polyvinylidene difluoride (PVDF) membrane (Millipore, Watford, UK) and blocked in PBST and 5 (wv) dried skimmed milk. The blots have been then probed in PBST plus milk with mouse polyclonal anti HIF1 (R D Biosciences, Abingdon, UK) diluted 1:500, rabbit polyclonal antiPHD2 (Abcam, Cambridge, UK) diluted 1:1000 and rabbit polyclonal antitubulin (Abcam) diluted 1:5000. To detect HIF1, the blot was incubated for four h at space temperature; to detect PHD2 and tubulin incubation was for 1 h at space temperature. Subsequently, every single blot was probed for 1 h at space temperature with peroxidase conjugated goat antimouse secondary antibody diluted 1:6000 (for HIF1) or goat antirabbit secondary antibody diluted 1:4000 (for PHD2 and tubulin). Antibody bound to protein was detected by enhanced chemiluminescence (ECL) kit (Amersham, GE Healthcare Life Sciences, Tiny Chalfont, UK) as per the manufacturer's protocol. Protein quantification was carried out utilizing GBoxHR Gel Doc technique (Syngene, Cambridge, UK).NAN 2015; 41: 201MicroRNA profiling of fibroblastsTotal RNA was extracted from 6 SALS and six control T75 fibroblast culture flasks making use of the mirVana Ations in about 500 HGSC samples [2. By means of computational analysis of TCGA data] isolation kit (Applied Biosystems) based on the manufacture's protocol. TaqManLow Density Arrays (TLDAs) had been utilized to measure the expression amount of 377 microRNAs (miRNAs) (Applied Biosystems).

ดึงข้อมูลจาก "https://wiki.surinsanghasociety.com/index.php?title=หน้าหลัก&oldid=5257"