Molecular Biology Related Protocols

Molecular Biology Related Protocols

Similar as that listed under the multiplex RT PCR protocol, except for the use of OligodT primers. It can also be beneficial to use a more processive ...
www.uchsc.edu/misc/diabetes/derc/prot_molecular.htm

Mathematical model for relative quantification in RT PCR

[PDF] A new mathematical model for relative quantification in real-time ...File Format: PDF/Adobe Acrobat
fication types in real-time RT-PCR are possible. (i) A relative .... protocol was used: denaturation program (95°C for 10 min), ...
www.umass.edu/research/genomics/files/CompQuant-Pfaffl.pdf

RT-PCR Procdure

[PDF] RT-PCR ProcdureFile Format: PDF/Adobe Acrobat - View as HTML
ThermoScript RT. 1ul. 20ul. Total. 8ul. 160ul. Add 8ul of the Master Mix to each sample. 50°C for 30 min and 85°C for 5 min in PCR machine. Store at -20°C.
www.ucsf.edu/xinlab/Protocol/RT-PCR%20Procedure.pdf

Real-Time RT-PCR Analysis of Carotenoid Genes

[PDF] Real-Time RT-PCR Analysis of Carotenoid Genes in Daucus carota L.File Format: PDF/Adobe Acrobat - View as HTML
normalizers for the Real-time RT PCR data. The expression of .... according to manufacturer's protocol. NanoDrop® ND-1000. spectrophotometer ...
www.wisc.edu/cbe/srp-bio/pdf/BautistaCarevised.pdf

Introduction to RNA Quantification and PCR

Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial distribution of RNA expression. A number of widely used procedures exist for detecting and determining the abundance of a particular mRNA in a total RNA sample. The most popular methods are: Northern blot analysis, Nuclease Protection Assays (NPA), in situ hybridization, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

In theory, each of these techniques can be used to detect specific RNAs and to determine their expression level. However, each method has inherent advantages and/or limitations. In general, Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to simultaneously examine multiple messages. In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression. Northern blotting and nuclease protection assays are also limited by their sensitivity. Ribonuclease protection assay is the most sensitive non-PCR based mRNA detection and quantitation procedure but RT-PCR is significantly more sensitive than this; it is capable of detecting moderately expressed transcripts from a single cell.

Full Details here

http://209.85.135.104/custom?q=cache:Y9dGBlb-qDYJ:www.uic.edu/classes/phyb/phyb569/2001/carlos.doc+RT+PCR+Protocol&hl=en&ct=clnk&cd=7&client=pub-9374796234212164

Total RNA preparations for RT-PCR

PDF] Single fly total RNA preparations for RT-PCR. Bertucci, Lisa A ...

File Format: PDF/Adobe Acrobat - View as HTML expression profiles by RT-PCR. Such RNA would need to be free of contaminating genomic DNA. Here, we present a variant of the Ambion RNAwiz protocol that we ... www.ou.edu/journals/dis/DIS84/Tec2%20Bertucci/Bertucci.pdf

Protocol Using QIAGEN OneStep RT-PCR Kit

[PDF] Protocol Using QIAGEN OneStep RT-PCR KitFile Format: PDF/Adobe Acrobat
This protocol serves as a guideline for one-step RT-PCR. .... QIAGEN OneStep RT-PCR. Protocol. Table 2. Thermal cycler conditions. Additional comments ...
www.uark.edu/ua/henrylab/Links/biochemgen/QIAGEN%20RT-PCR%20protocol.pdf

Quantitative Reverse-Transcription PCR (q-RT-PCR)

[PDF] Quantitative Reverse-Transcription PCR (q-RT-PCR)
File Format: PDF/Adobe Acrobat - View as HTML
Quantitative Reverse-Transcription PCR (q-RT-PCR). Adapted from Applied Biosystems protocol. Reagents. Trizol Reagent (Invitrogen #15596-026) ...
www.utexas.edu/research/sissonlab/protocols/molecularbiology/q-RT-PCR.pdf

IMMUNO PCR PROTOCOL

[DOC] IMMUNO PCR PROTOCOLFile Format: Microsoft Word - View as HTML
IMMUNO PCR PROTOCOL as. Obtaining cell ... autoclaved) to bottom of tube, spin down, and remove supernatent for RT-PCR; let beads/cell stand for 15 minutes. ...
www.dartmouth.edu/~yehlab/protocols/Immuno%20PCR/immuno1%20oligo.metho.doc

Method for real time quantitative RT-PCR

Genome Res. 6:995-1001, 1996
©1996 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051

A novel method for real time quantitative RT- PCR.

U E Gibson, C A Heid, and P M Williams

Genentech, Inc., South San Francisco, California 94080-4990, USA.

Abstract

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT- PCR ) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT- PCR.

Full Details Here
http://www.genome.org/cgi/content/abstract/6/10/995

Analysis of Relative Gene Expression by Real-Time PCR

Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 22DDCT Method
Kenneth J. Livak* and Thomas D. Schmittgen†,1
*Applied Biosystems, Foster City, California 94404; and †Department of Pharmaceutical Sciences, College of Pharmacy,
Washington State University, Pullman, Washington 99164-6534

Full Details here
http://www.umich.edu/~caparray/Files/LeivakddCt_paper.pdf

Real time quantitative PCR

Real time quantitative PCR.

C A Heid, J Stevens, K J Livak, and P M Williams

BioAnalytical Technology Department, Genentech, Inc., South San Francisco, California 94080, USA.

Abstract

We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post- PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.

Full Article Here
http://www.genome.org/cgi/content/abstract/6/10/986

Quantitative Real-Time PCR

Validation of Microarray Results Using Quantitative Real-Time PCR (QRT PCR) with Omniscript RT

Last Updated 04-01-2005

RNA used for quantitative real-time PCR should be identical to the RNA used on the microarray. The following protocol is slightly modified from the Direct Cy-Labeling Protocol Using Omniscript Reverse Transcriptase listed on our microarray website.

Full Article Here
http://omrf.ouhsc.edu/~frank/M_QPCR-OMNISCRIPT-RT.html

quantification of mRNA using real-time PCR

Absolute quantification of mRNA using real-time reverse
transcription polymerase chain reaction assays
S A Bustin
Academic Department of Surgery, St Bartholomew's and the Royal London School of Medicine and
Dentistry, Queen Mary and Westfield College, London E1 1BB, UK; Email: s.a.bustin@mds.qmw.ac.uk
ABSTRACT
The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained
from limited tissue samples. However, it is a complex....

Full Protocol Here
http://www.gene-quantification.de/bustin-2000.pdf

Real-Time PCR Quantitation

Real-Time PCR Quantitation The ability to monitor the real-time progress of the PCR completely revolutionizes the way one approaches PCR-based quantification of DNA and RNA. Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid targ.....

Full Protocol Here
http://dna-9.int-med.uiowa.edu/realtime.htm#rt%20quant

Primer and Probe Design

Primer and Probe Design
Primer Express software uses a set of default parameters to automatically select primer and probe sets. A summary of the primer and probe design guidelines is shown in Table 1. Even though no probe is required for SYBR Green I dye detection, it is still a good idea to use Primer Express software to select a primer and probe set when designing a SYBR Green I assay. Although no probe will be used, the primers will meet all the.....

Full Details Here
http://dna-9.int-med.uiowa.edu/realtime.htm#design

Real-Time or kinetic PCR

The DNA Facility houses the "real-time" or kinetic PCR instrument, the Applied Biosystems Model 7700 sequence detection system (the TaqMan instrument). The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. As little as a single copy of a particular sequence can be specifically amplified and detected. Theoretically, there is a quantitative relationship between amount of starting tar.....

Full Details Here
http://dna-9.int-med.uiowa.edu/realtime.htm

REAL-TIME PCR

REAL-TIME PCR

M. Tevfik Dorak, MD, PhD
 
Dorak MT (Ed): Real-Time PCR (Advanced Methods Series). Oxford: Taylor & Francis, 2006
Real-time reverse-transcriptase (RT) PCR quantitates the initial amount of the template most specifically, sensitively and reproducibly, and is a preferable alternative to other forms of quantitative RT-PCR that detect the amount of final amplified product at the end-point 1 2 (Freeman, 1999; Raeymaekers, 2000). Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (ie, in real time) as opposed to the endpoint detection 3,4 (Higuchi, 1992; Higuchi, 1993).

Full Protocol Here
http://dorakmt.tripod.com/genetics/realtime.html