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

Multiplex PCR: Critical Parameters and Step-by-Step Protocol

Multiplex PCR: Critical Parameters and Step-by-Step Protocol
BioTechniques 23:504-511 (September 1997)

O. Henegariu, N.A. Heerema, S.R. Dlouhy, G.H. Vance and
P.H. Vogt1
Indiana University, Indianapolis, IN, USA and 1Heidelberg
University, Heidelberg, Germany

ABSTRACT
By simultaneously amplifying more than one locus in the same reaction, multiplex
PCR is becoming a rapid and convenient screening assay in both the clinical and the
research laboratory. While numerous papers and manuals discuss in detail conditions
influencing the quality of PCR in general, relatively little has been published
about the important experimental factors and the common difficulties frequently encountered
with multiplex PCR. We have examined various conditions of the multiplex
PCR, using a large number of primer pairs. Especially important for a successful multiplex
PCR assay are the relative concentrations of the primers at the various loci, the
concentration of the PCR buffer, the cycling temperatures and the balance between the
magnesium chloride and deoxynucleotide concentrations. Based on our experience,
we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome
commonly encountered problems.

http://www.protocol-online.org/cgi-bin/prot/jump.cgi?ID=3151

PCR and multiplex PCR guide

Generalities
Components, PCR cycle, vials

Annealing time and temperature

Choosing PCR primers
How to design the primers

Extension time and temperature

Reaction volumes
Do they influence the results?

DNA template concentration

Number of PCR products
Multiplexing. How ?

Taq polymerase(s)
Comparisons and concentration

Primer amount
How much primer?

dNTP concentration
Interaction with magnesium

PCR buffers
Comparisons and concentration

MgCl2 concentration
Interaction with dNTP and salt (PCR buffer)

Salt (KCl) concentration
Essential optimization factor

Gel electrophoresis
Agarose and polyacrylamide (PAA)

Designing PCR programs
Customize them for your purpose

Adjuvants in PCR
DMSO, glycerol and BSA

Bisulfite Conversion Kit

methylSEQr™ Bisulfite Conversion Kit converts non-methylated cytosines (C) in your DNA sample to uracil (U). This kit is the first step in a three step workflow (step two is PCR amplification; step three is sequence analysis) that enables you to determine which cytosines in your sample are methylated by comparing the sequences of treated vs. non-treated DNA.
This kit enables you to:

Convert all cytosines (C) in your DNA sample to uracil (U).

Recover all of your sample DNA.

Minimize DNA degradation for optimum PCR amplification and sequence analysis.
Reliable Chemistry
The methylSEQr™ Bisulfite Conversion Kit takes advantage of the different sensitivity of cytosine and methylated cytosine to deamination by bisulfite, in which non-methylated cytosine is converted to uracil and methylated cytosine remains unreactive.
Fast Three-Step Protocol
methylSEQr™ Bisulfite Conversion Kit contains everything you need for a quick and easy three-step protocol:

1. Denature your DNA sample with methylSEQr™ Denaturation Buffer.
2. Convert unmethylated cytosines to uracils using methylSEQr™ Conversion Reagent.
3. Purify your treated DNA sample by centrifugation in a methylSEQr™ Column.

Step One of a Three-Step Methylation Analysis Workflow
There are three main steps in analyzing the methylation of your DNA sample:

1. Bisulfite Conversion with methylSEQr™ Bisulfite Conversion Kit
   Convert all non-methylated cytosines in your DNA sample to uracils.
2. PCR Amplification
   Design primers for PCR using Methyl Primer Express® Software
   Amplify both the treated and non-treated (control) DNA. During amplification, the uracils in your treated samples convert to thymines (T). To find thermal cyclers, PCR enzymes and kits, and products for PCR reaction purification, go to the PCR section of our catalog.
3. Sequence Analysis
   To detect which cytosines in your sample are methylated, you must compare the sequences of your treated vs. non-treated (control) DNA samples. You have many choices for sequence analysis:
   1. Go to the DNA Sequencing section of our catalog.
   2. Go to the SNP Genotyping section of our catalog.

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Note: See user's manual or package insert for limited label license, and trademark information. For Research Use Only. Not for use in diagnostics procedures.

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https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=602644&tab=DetailInfo

Bisulfite Conversion and Sequencing of Methylated DNA

methylSEQr™ System: Bisulfite Conversion and Sequencing of Methylated DNA
Applied Biosystems

DNA methylation patterns—are often considered as a second code, an additional layer of information superimposed on the DNA code that determines many phenotypic attributes. The DNA code is stable, but DNA methylation patterns change in response to spatial, temporal and environmental cues. After PCR amplification, the sequence of the bisulfite converted DNA will have C residues only if the C was methylated and will occur adjacent to G's (CpG). A...

http://www.biotechniques.com/default.asp?page=protocol&subsection=article_display&id=112346

EpiTect Bisulfite Kits For complete bisulfite conversion and cleanup of DNA for methylation analysis Streamlined 6-hour procedures for fast and reliable results Conversion rate of ≥99% using up to 2 μg template Unique DNA protection system which limits DNA degradation Optimized protocols including conversion of DNA from FFPE tissue Spin-column and 96-well plate formats for all throughput requirements

EpiTect Bisulfite Kits Links Protocols/Handbook MSDS Brochure

The QIAcube can automate this kit - find out more

Show detailed information

Bisulfite-PCR For Restriction Analysis And/or Sequencing

Bisulfite-PCR For Restriction Analysis And/or Sequencing

Source:
Jean-Pierre Issa, The University of Texas M. D. Anderson Cancer Center, Houstan

Bisulfite-PCR followed by restriction is a rapid and semi-quantitative method of analyzing DNA methylation. The PCR products are also suitable for either direct sequencing or cloning and sequencing. The most important step here is primer selection.

http://www.biocompare.com/jumpres.asp?type=protocol&protocolid=1891&siteid=30

Methylation Specific PCR Protocol

Methylation-Specific PCR

Protocol written by James Herman*

Methylation-specific PCR (MSP) is a simple rapid and inexpensive method to determine the methylation status of CpG islands. This approach allows the determination of methylation patterns from very small samples of DNA, including those obtained from paraffin-embedded samples, and can be used in the study of abnormally methylated CpG islands in neoplasia, in studies of imprinted genes, and in studies of human tumors for clonality by studying genes inactivated on the X chromosome.

http://www.mdanderson.org/departments/methylation/display.cfm?id=0CB11381-59AE-4D90-AC0B343F2D646EDD&method=displayFull

Protocol-Methylated CpG Island Amplification

Methylated CpG Island Amplification

Protocol written by Minoru Toyota

2. Materials

2.1. MCA

• Restriction enzymes SmaI, XmaI
• T4 DNA ligase
• Taq DNA polymerase
• 10X PCR reaction buffer:

http://www.mdanderson.org/departments/methylation/display.cfm?id=8A198378-986B-41FD-8778FA1399FBAB4D&method=displayFull

Single Nucleotide Primer Extension Protocol

DNA methylation analysis using Single Nucleotide Primer Extension (SNuPE)

Single Nucleotide Primer Extension is a powerful method which can be used for the precise analysis of methylation in a certain position. The procedure is shown on the figure. You treat your DNA with bisulphite and then anneal a primer which ends immediately before the site of analysis.

http://www.methods.info/Methods/DNA_methylation/SNuPE.html

SNuPE PCR Protocol

SNuPE Protocol

Reminder ­ Wear Gloves, lab coat and eye protection!!!

1. Make sure that there is enough dCTP and TTP (or dGTP and dATP) in the hot room
2. Set up your PCR master mix. Remember that you need to do a reaction for dCTP and TTP for each sample

http://www.genetics.ucla.edu/labs/fan/Protocols_SNuPE.htm