Quantifying nucleic acids by real-time PCR

Next workshop: date 4th & 5th September 2013

Earlybird registration (before 29th July deadline):

£460 academic participants, £560 other participants

After this deadline an additional £100 fee applies.

This two-day workshop is aimed at those who would like to use real-time PCR to quantify DNA or mRNA. It is based upon previous workshops we have run over the past nine years for the BBSRC Molecular Training for Industry programme, King's College graduate school and The Physiological Society (to view comments from previous attendees click here...). Participants are expected to have a theoretical understanding of the polymerase chain reaction and reverse transcription. No prior knowledge of real-time PCR is expected.

The workshop is held in newly-refurbished, modern teaching laboratories at Birkbeck, University of London. Birkbeck is located in the heart of Bloomsbury, close to Euston and King's Cross main line stations, and is served by most underground lines. For maps and travel information, click here...

Accommodation and travel costs are not included in the price. For a list of local hotels click here...

For the Terms and Conditions of registration,and information on payment methods, click here...

To register for this course click here... (you will be asked to confirm that you have read the Terms and Conditions before registering)

Course content

Workshop Day 1

Sample/template preparation

Theory: Isolation of DNA, total RNA and mRNA. Assessing template quantity and quality prior to qPCR.

Practical: Isolation of total RNA from tissue using spin columns. Analysis of total RNA quantity using a Qubit fluorimeter, and RNA quality using an Agilent Bioanalyzer.

Reverse Transcription (RT) of mRNA and preparation of known copy number standards

Theory: Use of total RNA or mRNA. RT priming strategies and enzymes.

Practical: Set up RT reaction with total RNA isolated earlier. Set up a qPCR assay with the newly-synthesised cDNA. Check PCR product specificity/identity by agarose gel electrophoresis.

Assay Design

Theory: qPCR assay development; primer design; product detection strategies; PCR conditions/optimisation.

Standard Operating Procedures

Theory: How to avoid contamination

Workshop Day 2

qPCR

Practical: Set up and run a quantititive probe assay. Prepare a standard dilution series. Set up and run a quantitative SYBR Green assay with standards, cDNA samples, and positive and negative controls.

Data analysis

Practical: Evaluate assay efficiency, sensitivity, reliability, accuracy. Compare results from probe and SYBR assays. Analyse data using absolute and comparative methods with correction for qPCR efficiency.

Theory: Aspects of data analysis: Normalisation of qPCR data - demonstration of geNorm, absolute versus relative quantification, quality control and MIQE. Troubleshooting. Examples of applications.