RNA extraction and quality analysis of extracted RNA

RNA analysis is one of the most efficient ways to get a real view of metabolic activities in a biotope. However, this extraction is often a limiting step in the procedure, especially on complex environmental samples. This extraction can be problematic in terms of the amount of material recovered as well as the quality of this material.

The Plinius Cursus offers the workshop “RNA extraction and quality analysis of extracted RNA”. In this module, we propose to perform an RNA extraction from complex samples (sediments and filter from a water sample). After cell lysis, the RNAs will be purified and processed to clean them of any residual DNA. The quality of these RNAs will be checked on Agilent’s BioAnalyzer. After a reverse transcription step, a qPCR will then be performed to quantify the cDNAs but also to verify in another way the absence of DNA in the extracted RNAs.

At the end of the workshop, the PhD student will have addressed several points: the critical steps of an RNA extraction, the various possible extraction protocols, how to evaluate the quality and quantity of the RNAs, how to exploit them.

Timeline

The workshop will take place during 2 days

1. Day 1 morning: Theoretical introduction. RNA extraction on sediment samples (RNeasy Power Soil total RNA kit, QIAGEN)

2. Day 1 afternoon: RNA extraction from the filter (water samples, RNeasy Power Water kit, QIAGEN) and treatment (twice) with turbo DNase (Invitrogen) of all samples. Quantification of the extracted RNAs using the nanodrop.

3. Day 2 morning: Preparation of the chip for the quality analysis of the extracted RNAs on Bioanalyzer (Agilent) – RNA reverse transcription to synthesize cDNAs using random-qPCR primers for quality control of the extracted RNAs and quantification of the cDNAs obtained.

4. Day 2 afternoon: analysis of results

Students prerequisites

• qPCR theory will not be explained here but will be considered as already mastered.
• Agreement of the thesis director