Navigation Links
Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ Real-Time PCR Detection System and iQ Supermix, Rev A

ash-frozen tissue in a 1 ml microcentrifuge tube and vigorously homogenized for 30 sec using an Ultraturrax T8 homogenizer (Ika Laboratories). Total RNA was DNasetreated using DNA-free reagent (Ambion), and RNA yields were determined by fluorescence at 530 nm using RiboGreen RNA quantitation reagent (Molecular Probes) as described by the manufacturer. First-strand cDNAs were synthesized from equal amounts of total RNA (1 g/reaction) using oligo(dT) primers and SuperScript II reverse transcriptase (Invitrogen) according to the manufacturers instructions.

Gene-specific primers (GSPs) were designed to anneal near the 3' end of two mRNA transcripts and to generate PCR products 75300 base pairs long. Three GSP pairs amplify different overlapping regions of a single transcript that is enriched in the macula, while one GSP pair detects a human housekeeping gene transcript, β-actin (ACTB), which is constitutively expressed in the neural retina. The amplified regions spanned exon-exon junctions when possible. All primers were purchased from Proligo. RT-PCR was performed using the GSP pairs in reactions amplifying across a gradient of annealing temperatures to identify optimal reaction conditions for real-time RT-PCR, and PCR product lengths were verified on a 4.5% Super AcrylAgarose gel (DNA Technologies). Real-time quantitative RT-PCR was performed using an iCycler iQ system (Bio-Rad). The rate of accumulation of amplified DNA was measured by continuous monitoring of SYBR Green I (Molecular Probes) fluorescence. Melt curves of the reaction products were generated, and fluorescence data were collected at a temperature above the melting temperature of nonspecific products (Morrison et al. 1998).
'"/>

Source:


Page: All 1 2 3 4 5 6 7 8

Related biology technology :

1. Rapid, High Throughput Mutation Discovery Using Fluorescent SSCP on the ABI PRISM 3100 and 3100Avant Genetic Analyzers
2. Detection of Genetically Modified Soybean in Processed Foods Using Real-Time Quantitative PCR with SYBR Green I Dye on the DNA Engine Opticon 2 System
3. Monitoring Microbial Populations Using Real-Time qPCR on the MJ Research Opticon 2 System
4. Rapid SNP Genotyping of the Pharmacogenomically Important Allele CYP2D6*4 Using Real-Time qPCR on the DNA Engine Opticon 2 System
5. Real-Time Multiplex PCR from Genomic DNA Using the iCycler iQ Detection System
6. Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A
7. Real-Time Quantification of Genomic DNA Using DyNAzyme II DNA Polymerase and SYBR Green I Dye
8. Quantitation of Lymphangiogenesis Using the iCycler iQ Real-Time PCR Detection System and Scorpions Detection System, Rev A
9. Real-Time Immuno-PCR on the iCycler iQ System, Rev A
10. Real-Time PCR/Melt-Curve Analysis: SNP Detection With FRET, Rev A
11. Real-Time PCR: General Considerations, Rev A
Post Your Comments:
(Date:11/18/2014)... CITY , Nov. 18, 2014  Great Basin Scientific, ... today announced that the management will host a conference call ... and development programs on Thursday, Nov. 20, at 4:30 pm ... we have made since the Company,s recent IPO and would ... Great Basin,s management," said Ryan Ashton , President and ...
(Date:11/17/2014)... , Nov. 17, 2014 Soligenix, ... late-stage biopharmaceutical company developing products that address unmet ... and biodefense, announced today the formation of a ... guidance to the Company as it advances the ... for the treatment of cutaneous T-cell lymphoma (CTCL), ...
(Date:11/15/2014)... Collaborative Consulting, a privately held, employee-based ... of the Boston Globe’s “Top Places to Work 2014”. ... Massachusetts to receive the honor. , The Boston Globe’s ... of their most valuable resource: the people who work ... are based on an array of criteria, including the ...
(Date:11/15/2014)... November 14, 2014 The ... announced today that the society’s Governing Board has ... Edwards Lifesciences chief scientific officer and Dr. Charles ... officer, Abbott Vascular. Mr. Rowe and Dr. ... device development experts. , Dr. Nabil Dib, ...
Breaking Biology Technology:Great Basin Scientific Announces Conference Call to Provide Update on Corporate Progress 2Soligenix Announces Formation of Cutaneous T-Cell Lymphoma Medical Advisory Board 2Soligenix Announces Formation of Cutaneous T-Cell Lymphoma Medical Advisory Board 3Soligenix Announces Formation of Cutaneous T-Cell Lymphoma Medical Advisory Board 4Soligenix Announces Formation of Cutaneous T-Cell Lymphoma Medical Advisory Board 5Soligenix Announces Formation of Cutaneous T-Cell Lymphoma Medical Advisory Board 6Collaborative Consulting Honored with Boston Globe Top Places to Work 2014 2International Society for Cardiovascular Translational Research Governing Board Adds Medical Industry Leaders 2
... Group in North America with this Elevated Partner ... ... an Oracle System,Integrator, today announced it has achieved Oracle Certified Advantage,Partner status ... the highest level in the Oracle PartnerNetwork -- BIAS had,to demonstrate superior ...
... for the Long-Term Care Channel, BASKING ... wholly owned subsidiary of Millennium Biotechnologies,Group, Inc. ... the initial,$200,000.00 monthly shipment from Millennium Biotechnologies, ... the previously announced $20,000,000,Five- year exclusive Purchase ...
... Life Sciences R&D to ... Ensure Continued Growth, ... Authority (EDA) announced today that Tris Pharma, Inc. will,expand its ... bonds issued under the state,s Edison Innovation,Fund. Representatives from the ...
Cached Biology Technology:BIAS Achieves Certified Advantage Partner Level in Oracle PartnerNetwork 2BIAS Achieves Certified Advantage Partner Level in Oracle PartnerNetwork 3Millennium Ships and Records the First $200,000 of Revenue Generated From the Five-Year $20,000,000 Exclusive Purchase Order 2Millennium Ships and Records the First $200,000 of Revenue Generated From the Five-Year $20,000,000 Exclusive Purchase Order 3Edison Innovation Fund Helps Tris Pharma Expand 2Edison Innovation Fund Helps Tris Pharma Expand 3
(Date:11/7/2014)... Debra Auguste, associate professor, biomedical engineering, in the ... of New York, have identified a molecule that ... the most aggressive forms of breast cancer. , ... mortality rate owing to aggressive proliferation and metastasis ... Professor Auguste,s team, discovered the overexpression of intercellular ...
(Date:11/6/2014)... yet progress in preventing and treating migratory cancer ... challenging to design drugs that work against metastasis," ... at Harvard Medical School. , "Unfortunately, many ... , Gujral and colleagues have now identified a ... how metastasis begins. Their findings may also inform ...
(Date:11/5/2014)... RENO – Kelly Redmond has dedicated his career ... knowledge to a general audience. , As deputy ... Climate Center at Nevada,s Desert Research Institute, Redmond ... the management, application and dissemination of climate data ... Annual Fall Meeting in San Francisco on December ...
Breaking Biology News(10 mins):Migration negation 2Migration negation 3Nevada climatologist to be honored at AGU Fall Meeting for lifetime of public outreach 2
... position is a determinant of antiretroviral treatment effectiveness during ... even among subjects with high rates of drug adherence, ... issue of the Journal of Psychosomatic Research. The ... Harvard School of Public Health (HSPH), Massachusetts General Hospital, ...
... of rotten meat on the premises of a wholesaler in ... grocery stores everywhere is not to be underestimated, the importance ... reliable measurement methods that enable large numbers of samples to ... place in order to make comprehensive monitoring a reality. In ...
... of the GSF National Research Center for Environment ... Centres) the group is continuing to search for cellular ... a prion infection. In collaboration with colleagues from the ... the group used micro-array technologies micro-arrays are ...
Cached Biology News:Socioeconomic position associated with effectiveness of HIV drugs 2New method reveals substances on surfaces of any kind 2Prions and retroviruses -- an unholy alliance? 2
Mouse monoclonal antibody raised against a partial recombinant PASK. NCBI Entrez Gene ID = PASK...
Agarose II (Low Melt)...
...
... I can be used in intracellular staining ... cells and to serve as an antibody ... cell permeabilization is a reversible process, it ... the presence of saponin during intracellular staining. ...
Biology Products: