isolation cdna synthesis and real time pcr

... to ensure the quality of RNA and cDNA PCR products amplified from cDNA are indicated by asterisks Sequences for the cDNAs were confirmed by cloning and sequencing PCR products at higher molecular ... (miR159a and miR159b) have been sequenced more than 5,000 times in mature pollen Since the expression of these two miRNAs was not detected by either real- time PCR or regular RT -PCR (Table and Figure ... Universal PCR master mix (Applied Biosystems, Foster City, CA), μl of each primers (900 nM), μl Taqman probe (250 nM), and 1.5 μl cDNA sample All real- time PCR reactions were performed in a StepOne real- time...

Ngày tải lên: 12/08/2014, 03:20

... cells The real- time PCR (RT -PCR) technology utilizes the standard PCR method with the addition of measuring the accumulation of amplified DNA in real- time by a fluorescent signal RT -PCR uses the ... assays, RNA extraction and realtime PCR analysis NJ drafted and wrote the manuscript MG developed the protocol for the binding assay, radiolabeled and purified the clinical isolate and performed the ... single-tube real- time reverse transcription -PCR assay J Clin Microbiol 2001, 39:4093-4096 Mohamed N, Elfaitouri A, Fohlman J, Friman G, Blomberg J: A sensitive and quantitative single-tube real- time...

Ngày tải lên: 12/08/2014, 04:21

... gene synthesis, including the introduction of bioinformatics software, oligonucleotide synthesis, LCR based gene synthesis, PCR based gene synthesis, real- time PCR, DNA purification/extraction and ... Top-Down PCR based gene synthesis 47 4.3 Experiment verification of TopDown one-step gene synthesis 49 4.3.1 4.3.2 Non-competition one-step real- time gene synthesis 49 4.3.3 One-step and ... Department of Electrical and Computer Engineering Towards automatic gene synthesis with bioinformatics software, novel one-step real- time PCR assembly, and lab-chip gene synthesis Huang Mo Chao...

Ngày tải lên: 14/09/2015, 08:48

... The kinetics difference between standard PCR amplification and onestep PCR gene synthesis has been analyzed using this model, and validated using real- time gene synthesis Analysis of the experimental ... Automatic TouchDown one-step real- time gene synthesis Automatic TouchDown (ATD) one-step process was optimized using real- time PCR conducted with Roche’s LightCycler 1.5 real- time thermal cycling machine ... single PCR from a pool of 62 oligonucleotides of nM 5.1.2 Mechanisms of PCR synthesis process In order to further understand the mechanisms of PCR synthesis process, the underlining kinetics of PCR...

Ngày tải lên: 14/09/2015, 08:48

... WI) cDNA synthesis cDNA was produced using the Superscript III RT -PCR System (Invitrogen, Rockville, MD) according to the manufacturer's recommendations for oligo(dT)20 primed cDNA- synthesis cDNA ... Toledo, VZV strain Schenke and HIV-1 strains BaL, NL4.3 and RFW were propagated according to standard protocols [16-20] The cell types infected, MOI, time of infection and percentage of cells infected ... (Toledo and Towne strains) and VZV (Schenke strain) and harvested at the earliest time of maximal productive infection as illustrated in Table The expression levels of 10 commonly used QPCR normalisation...

Ngày tải lên: 18/06/2014, 18:20

... design and coordination of the study and helped to draft the manuscript All authors read and approved the final manuscript Material and Methods Virus culture and virus detection by real- time PCR ... primed cDNA- synthesis cDNA synthesis was performed using µg of RNA, at 50°C Finally, cDNA was diluted 1:5 before use in QPCR Quantitative TaqMan PCR Primers, TaqMan probes and QPCR conditions for ... http://www.virologyj.com/content/2/1/7 The respective MOI and time of cell culture are shown in table and were chosen to allow maximal infection as determined by immunofluorescence and real- time PCR [8-11] For kinetic studies,...

Ngày tải lên: 18/06/2014, 22:20

... a PCR reaction containing approximately 100 copies of bacteriophage λ DNA and a primer pair directed against λ DNA The performance of the PCR was monitored by quantitative real- time PCR (qPCR) ... the target DNA, and hybridization to a fluorogenic probe are conducted in a single PCR and therefore the chances of possible contamination are minimised The main advantage of real- time detection ... fluid of these 27 HSV PCR positive subjects The distribution is left censored and the majority of subjects had

Ngày tải lên: 19/06/2014, 08:20

... employing microtiter and transwell plates Semiquantitative polymerase chain reaction (PCR) and quantitative real- time PCR have been recently developed and employed for the detection and quantitation ... Figure A – C: Analysis of HA and quantitative real- time PCR data employed for the determination of JC viral load A – C: Analysis of HA and quantitative real- time PCR data employed for the determination ... that real- time PCR is a sensitive and reliable method for in vitro quantitation of JCV In vitro measurement of JCV DNA levels was highly reproducible over a large dynamic range, and real- time PCR...

Ngày tải lên: 19/06/2014, 08:20

... WI) cDNA synthesis cDNA was produced using the Superscript III RT -PCR System (Invitrogen, Rockville, MD) according to the manufacturer's recommendations for oligo(dT)20 primed cDNA- synthesis cDNA ... Toledo, VZV strain Schenke and HIV-1 strains BaL, NL4.3 and RFW were propagated according to standard protocols [16-20] The cell types infected, MOI, time of infection and percentage of cells infected ... (Toledo and Towne strains) and VZV (Schenke strain) and harvested at the earliest time of maximal productive infection as illustrated in Table The expression levels of 10 commonly used QPCR normalisation...

Ngày tải lên: 20/06/2014, 01:20

... design and coordination of the study and helped to draft the manuscript All authors read and approved the final manuscript Material and Methods Virus culture and virus detection by real- time PCR ... primed cDNA- synthesis cDNA synthesis was performed using µg of RNA, at 50°C Finally, cDNA was diluted 1:5 before use in QPCR Quantitative TaqMan PCR Primers, TaqMan probes and QPCR conditions for ... http://www.virologyj.com/content/2/1/7 The respective MOI and time of cell culture are shown in table and were chosen to allow maximal infection as determined by immunofluorescence and real- time PCR [8-11] For kinetic studies,...

Ngày tải lên: 20/06/2014, 04:20

... a PCR reaction containing approximately 100 copies of bacteriophage λ DNA and a primer pair directed against λ DNA The performance of the PCR was monitored by quantitative real- time PCR (qPCR) ... the target DNA, and hybridization to a fluorogenic probe are conducted in a single PCR and therefore the chances of possible contamination are minimised The main advantage of real- time detection ... fluid of these 27 HSV PCR positive subjects The distribution is left censored and the majority of subjects had

Ngày tải lên: 20/06/2014, 04:20

... employing microtiter and transwell plates Semiquantitative polymerase chain reaction (PCR) and quantitative real- time PCR have been recently developed and employed for the detection and quantitation ... Figure A – C: Analysis of HA and quantitative real- time PCR data employed for the determination of JC viral load A – C: Analysis of HA and quantitative real- time PCR data employed for the determination ... that real- time PCR is a sensitive and reliable method for in vitro quantitation of JCV In vitro measurement of JCV DNA levels was highly reproducible over a large dynamic range, and real- time PCR...

Ngày tải lên: 20/06/2014, 04:20

... Detection limit and standard curve of the multiplex real- time PCR The detection limit and standard curve of the multiplex real- time PCR was determined using S Typhimurium ATCC 14028 and S Enteritidis ... multiplex real- time PCR assay showed 100% specificity for Salmonella spp and S Typhimurium and 99.1% for S Enteritidis The multiplex real- time PCR assay showed 100% sensitivity for Salmonella spp and ... the results of the standard culture method of isolated field and reference strains with those of the multiplex real- time PCR Results Specificity of the multiplex real- time PCR A total of 128 bacterial...

Ngày tải lên: 07/08/2014, 23:22

... for positivity and negativity We determined the expression of E-cadherin, α-, β- and γcatenin in human breast carcinomas using real time quantitative polymerase chain reaction and investigated ... chain reaction The levels of E-cadherin, α-, β- and γ-catenin transcripts from the prepared cDNA were determined by real time quantitative RT -PCR, based on the Amplifluor™ technology, using the ... invasion and degradation of surrounding matrix, and migration into the vasculature and surrounding tissue, E-cadherin catenin complex is reintroduced and cells adhere to the vasculature and form...

Ngày tải lên: 09/08/2014, 07:21

... by real- time PCR of Page of which two were positive by pp65 antigenemia assay and 12 were negative by both real- time PCR and pp65 antigenemia assay One donor was positive only by realtime PCR ... antigenemia and real - time PCR The highest copy number detected by real - time PCR was 29, 768 copies/ml and lowest was copies/ml The highest positive pp65 cell count recorded among them was 78 cells and ... (chi-square test p = 0.037%) Correlation between pp65 and real time PCR results Among the 76 kidney donors 58 (73.68%) were positive for real time PCR and of the 56, 39 (51.0%) were positive for pp65...

Ngày tải lên: 12/08/2014, 02:20

... use Page of and PCV-2), and control (sterile water) were processed with the real- time PCR Reproducibility of real- time PCR To determine the reproducibility of the real- time PCR, the standard plasmid ... sensitivity of the real- time PCR is 100 times greater than that of the conventional PCR (Figure 1C) Specificity of the real- time PCR The real- time PCR gave positive results for the standard plasmid ... replicates from each of four times) were relatively small (Table 1) Detection of PPV in clinical samples by real- time PCR and conventional PCR Real- time PCR and conventional PCR were used simultaneously...

Ngày tải lên: 12/08/2014, 02:20

... were tested for specificity by RTPCR and also by qRT -PCR, followed by a dissociation curve and agarose gel electrophoresis Real- time PCR conditions and analysis PCR reactions were performed in ... ubiquitin and elongation factor-1 alpha For species with both sexual and apomictic reproductive mode, the best reference genes for qRT -PCR experiments have not been reported yet Real time PCR has ... quantitative real- time PCR Biochemical and Biophysical Research Communications 2006, 345(2):646-651 Reid K, Olsson N, Schlosser J, Peng F, Lund S: An optimized grapevine RNA isolation procedure and statistical...

Ngày tải lên: 12/08/2014, 03:20

... reaction containing: μL of cDNA, 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, La ... RPS13 CYP RPS13 SAND TUB SAND TUB EF1alpha SAND GAPDH RPS13 UBQ TUB UBQ UBQ EF1alpha TUB GAPDH TUB RAN1 RAN1 ACT SAND ACT GAPDH ACT SAND ACT SAND RAN1 CYP GAPDH TUB GAPDH GAPDH GAPDH SAND GAPDH GAPDH ... young, small leaves and stage C to fully expanded ones Three independent samples of each of the developmental stages of flowers and leaves were taken RNA isolation and cDNA synthesis Mitchell...

Ngày tải lên: 12/08/2014, 03:21

... nM HAPBprobe Cyan500-CGAGAATCACCCTGCCAGACTTCCGT-BBQ 100 nM Sybr Green Real Time PCR TaqMan Real time PCR reaction Real time PCR reactions that target the E1 region of HPV were performed using Roche ... contributions WTS and JW-C prepared the manuscript and developed and performed the PCR assays EA and MC identified and obtained oral cancer tissue EMK and SC-U and identified and obtained cervical ... genome of HPV6 and HPV11, respectively, were obtained from the American Type Culture Collection (ATCC) and were used to derive standard curves for HPV6 and 11 in the real time PCR assay Seaman...

Ngày tải lên: 12/08/2014, 04:20

... higher specificity, and easy standardization Thus, quantitative RT -PCR assay might eventually replace virus isolation and conventional reverse transcription -PCR as the new gold standard for the rapid ... sequence-specific primer design and require RNA isolation procedures which make it expensive and labour intensive Recently, several studies have illustrated the application of real- time (RT) PCR technology or ... detection and quantification The additional advantage of utilizing SYBR Greenbased real- time RT -PCR is that it is relatively easy to design and test primer pairs that are suitable for RT -PCR analysis...

Ngày tải lên: 12/08/2014, 04:21