cdna synthesis for real time pcr

... electrophoresis Real-time PCR conditions and analysis PCR reactions were performed in 96-well plates with the Chromo4 Real-Time PCR Detector System (BioRad®) using SYBR® Green to detect dsDNA synthesis ... elongation factor-1 alpha For species with both sex-ual and apomictic reproductive mode, the best reference genes for qRT-PCR experiments have not been reported yet Real time PCR has been done to ... Aliquots from the same sscDNA sample were used with all primer sets in two separate experiments Two biological replicates for each of the 20 samples were used for real-time PCR analysis, and three...

Ngày tải lên: 12/08/2014, 03:20

... reaction containing: 2 μL of cDNA, 2 mM MgCl2, 0.2 mM each dNTP, 0.4 μL of each primer and 1.25 U enzyme Real-time PCR Mitchell Real-time PCR was performed in an Mx 3005P QPCR system (Stratagene, ... results in real-time PCR Nucl Acids Res 2002, 30(9):e36. 4 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe AFS: Accurate normalization of realtime quantitative RT-PCR data by ... equivalent ofcDNA and 1 pM gene-specific primers (Tab 3) was subjected to the following protocol: 95°C for 10 min fol-lowed by 50 cycles of 95°C for 30 sec, 60°C for 1 min and 72°C for 30 sec,...

Ngày tải lên: 12/08/2014, 03:21

... information for selecting real-time RT-PCR reference genes, as was done for Arabidopsis using the large public collection of data from Affymetrix GeneChip experiments [12] Unfortu-nately, for ... in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization Reference genes used for normalization ... transcriptional analyses To date, a limited number of real-time RT-PCR experiments focusing on grape berries has been published Based on microarray and real-time RT-PCR data, UBQ-L40 [14] and one paralog of...

Ngày tải lên: 12/08/2014, 05:20

... Performance of TopDown one-step gene synthesis and its real-time analysis 51 4.4.1 Performance of TD one-step gene synthesis 51 4.4.2 Analysis of real-time gene synthesis 54 4.5 Discussion 58 ... oligonucleotide synthesis, LCR based gene synthesis, PCR based gene synthesis, real-time PCR, DNA purification/extraction and enzymatic error filtering Also, fundamentals of gene synthesis related ... for gene synthesis 49 4.3.2 Non-competition one-step real-time gene synthesis 49 4.3.3 One-step and two-step PCR-based gene synthesis 50 4.3.4 Agarose gel electrophoresis 51 4.4 Performance...

Ngày tải lên: 14/09/2015, 08:48

... process was optimized using real-time PCR conducted with Roche’s LightCycler 1.5 real-time thermal cycling machine with a temperature transition of 20°C/s Real-time gene synthesis was conducted with ... Schematic illustration of PCR-based gene synthesis One-step synthesis combines PCA and PCR amplification into a single stage The two-step synthesis is performed with separate stages for assembly and ... [110, 111] Trang 4In this study ATD one-step gene synthesis and real-time PCR are combined together to investigate the gene synthesis process for the promoter of human calcium-binding protein...

Ngày tải lên: 14/09/2015, 08:48

... therapy The real-time PCR method was developed for the identification of the specific mutations in the CYP2C19 gene (CYP2C19 *2 and CYP2C19*3) and ITGB3 gene (PlA1/ A2) The real-time PCR method ... for DNA extraction and Realtime Techniques - Genomic PCR and mutation analysis are time-saving methods: saving an average of 45 minutes for DNA extraction and 90 minutes for gene polymorphism ... Kit (Lytech, Russia) Realtime - PCR technique was used to identify polymorphism of CYP2C19 gene (CYP2C19*2 , CYP2C19*3) and ITGB3 gene (PlA1 / PlA2) The primers used for PCR of each polymorphism...

Ngày tải lên: 22/01/2020, 14:38

... from the real-time qPCR assay for COL11A1 variant A in tumor breast cDNA samples: five positive samples, two negative and a blank (green line) Fig 5 Results from the real-time qPCR assay for COL11A1 ... curve for the real-time qPCR detection of COL11A1 variant E: amplicons ranging from 5 × 10 6 -5 × 10 1 copies E/ μl serve as standards Fig 2 Representative standard curve for the real-time qPCR ... distinct 132 bp band in all PCR products Development, analytical and clinical evaluation of the real-time qPCR methodology for theCOL11A1 variants detection Real-time qPCR methodologies were developed...

Ngày tải lên: 22/09/2020, 23:49

... E Open AccessAnalytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded ... O’Donnell et al.: Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded ... Langland R, et al: Multisite Analytic Performance Studies of a Real-Time Polymerase Chain Reaction Assay for the Detection of BRAF V600E Mutations in Formalin-Fixed Paraffin-Embedded Tissue...

Ngày tải lên: 05/11/2020, 06:20

... AccessSelection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis Huiyun Song1,2,3, Wenmai Mao1,2,3, ... reference genes Reverse-transcribed cDNA from each sample was used as a template with primers for standard PCR amplifica-tion Electrophoresis verified all PCR products were specific with single ... suitable reference gene for specific conditions using RT-Fig 2 Average expression stability values (M) for 20 candidate reference genes calculated by geNorm Trang 7PCR is therefore very important...

Ngày tải lên: 28/06/2021, 20:42

... reference genes are listed in Table 3 Quantitative real-time polymerase chain reaction RT-PCR reactions were performed in a Roch lightCycler 96 Real-Time System using Power SYBR green master mix ... cycler at 25 °C for 10 min, followed by 42 °C for 60 min, and 85 °C for 5 min Samples were then cooled to 4 °C cDNA samples were stored at − 20 °C for RT-qPCR analysis Primer design Thirteen candidate ... Quantitative Real-Time PCR Experiments (MIQE) guidelines46, standard curves were generated for each candidate reference gene using the Ct value that resulted from duplicate serial dilutions of cDNA obtained...

Ngày tải lên: 19/11/2022, 11:42

... conditions for quantitative real-time PCR data normalization Frontiers in Plant Science 7 (2016). 10 Niu, X et al Selection of reliable reference genes for quantitative real-time PCR gene expression ... Pfaffl, M W Quantitative real-time RT-PCR–a perspective Journal of Molecular Endocrinology 34, 597–601 (2005). 40 Gachon, C., Mingam, A & Charrier, B Real-time PCR: what relevance to plant ... salt, and heavy metal stress conditions Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses...

Ngày tải lên: 04/12/2022, 16:32

... Trung Quốc Trong nghiên cứu này, chúng tôi xây dựng quy trình Real-time PCR nhằm phát hiện gạo chuyển gen CpTI Quy trình Real-time PCR với cặp mồi CpTI-F và CpTI-R, nồng độ mồi 300 nM, nồng độ ... thiết kế xong, mồi được tổng hợp bởi công ty Sigma Aldrich ở nồng độ 100 µM Real-time PCR Các phản ứng Real-time PCR được thực hiện trên máy Lightcycler96 của hãng Roche Tổng thể tích mỗi phản ... trình bày trong Bảng 2 Bảng 2 Chương trình khuếch đại phản ứng Real-time PCR Trang 4Biến tính 95 600 1 Trước đó, phản ứng Real-time PCR được tối ưu nhiệt độ bắt cặp từ 55 oC – 66,4 oC (gradient...

Ngày tải lên: 18/02/2023, 05:29

... (see Results – Multiplex real-time PCR ofMixed Agent and Mixed primer/probe Cocktails (prep for Set 2) for details) Further analysis of the real-time PCR data was per-formed by plotting the Ct ... probe lengths also presented with associated real-time PCR channels used for detection Table 4 Summary of limited multiplex real-time PCR results for individual agents Values in the cells indicate ... cost and time as compared to current real-time PCR based approaches Results Rationale for experimental approach Current biosurveillance strategies predominantly em-ploy singleplex real-time PCR assays...

Ngày tải lên: 28/02/2023, 07:54

... 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

... 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