Quantifying nucleic acids by real-time PCR – Practical Workshop
Next workshop: Thursday and Friday 23rd & 24th November 2017
£460 academic participants, £560 other participants
Earlybird registration deadline 27th October 2017. After this deadline an additional £100 fee applies.
Scroll down to see a summary of the feedback from the previous two years’ participants and workshop timetable.
|Register here for this hands-on course (you will be asked to confirm that you have read the Terms and Conditions before registering).This two-day workshop is aimed at those who would like to use real-time PCR to quantify DNA or mRNA. It has evolved from workshops we have run over the past 14 years for the BBSRC Molecular Training for Industry programme, King’s College graduate school and The Physiological Society. 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. Accommodation and travel costs are not included in the price. This workshop will be held at University College London, Gower Street. For more information on payment methods and how to get there please visit the links to the right.
Feedback summary for 2014 & 2015 (n=106)
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Workshop Day 1 – 09.45-17.30
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.
Theory: qPCR assay development; primer design; product detection strategies; PCR conditions/optimisation.
Standard operating procedures
Theory: How to avoid contamination
Workshop Day 2 – 09.15-16.30
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.
Practical: Evaluate assay efficiency, sensitivity, reliability, accuracy. Compare results from probe and SYBR assays.
Theory: Aspects of data analysis: Normalisation of qPCR data – demonstration of geNorm, absolute versus relative quantification, quality control and MIQE.
Practical: Analyse a provided RT-qPCR dataset. Evaluate assay efficiency, select reference genes for normalisation, normalise data and compare group means (a limited number of computers will be provided but we ask participants to bring a PC laptop if possible).
Theory: Troubleshooting and questions.