qPCR for monitoring prokaryotic biodiversity

Quantitative PCR is a technique whose use has been democratised. More and more research topics as well as diagnostic approaches are using qPCR.

The Plinius Cursus offers the workshop “qPCR for monitoring prokaryotic biodiversity”. In this workshop, we propose to familiarise you with this technique through the quantification of prokaryotic biodiversity in complex environments.

In this module, we propose to perform a DNA extraction from sediment samples. In the first part, we will approach qPCR through a more theoretical vision, during which we will also discuss the notions of biodiversity monitoring via the gene coding for RNA16S. The training will then be oriented towards learning the technique. After cell lysis, the DNA will be purified. The quality and quantity of these DNAs will be controlled via two approaches Qbit and Nanodrop. The qPCR will be performed in a Biorad CFX96 OPUS controlled by CFX Maestro software. After conceptualising the experiment, the students will perform the qPCR and analyse the data obtained to quantify the prokaryotic population of their sample via the use of a standard range.

At the end of the workshop, the PhD student will have covered several points: the critical steps of a DNA extraction, how to assess the quality and quantity of total DNA, how to quantify a population in terms of primers, protocols, how to perform a qPCR and how to analyse the results obtained.

Timeline

The workshop will take place during 2 days

1. Day 1 morning: Theoretical introduction (qPCR, biodiversity quantification

2. Day 1 afternoon: DNA extraction on sediment samples (DNeasy PowerSoil Pro Kit, QIAGEN). Quantification and control of extracted DNAs using the nanodrop.

3. Day 2 morning: Familiarisation with the CFX Maestro software (preparation of plate design and program), plate preparation, qPCR for the quantification of the gene coding for bacterial RNA16S in a complex DNA sample.

4. Day 2 afternoon: analysis of results

Students prerequisites

• This course can be used as a preamble to the RNA course as it provides the necessary skills for the implementation and interpretation of qPCR, a technique used but not explained in the RNA course.
• Agreement of the thesis director