It is essential to have a good grasp of the theoretical and technical aspects of Quantitative PCR in order to generate reliable and reproducible results. The platform offers several training modules allowing you to grasp and understand the different aspects of the technology in accordance with the MIQE recommendations (minimum required for the publication of a QPCR paper ) and thus acquire a good degree of autonomy in the management of your QPCR projects.

training modules offered (details below):

The fundamental modules A, B and D, organised several times a year, allow you to master QPCR technology expertly and independently. Announcements and registrations are made by email.

Other modules are available upon request ( bariza.blanquier@inserm.fr )

A- Quantitative PCR: Theoretical aspect

This section aims to present all the parameters of amplification and dissociation kinetics and to highlight their importance in quantification calculations.

1- Measurement of the fluorescence amplification signal:

Hybridization probes (Taqman and molecular beacon)

The sybergreen

2- Analysis of the dissociation curve (Melting Curve Analysis):

Definition of TM

Sequence specificity and TM of the amplicon

Contaminations

Primer dimers

PCR quality and validation

3- Study of amplification kinetics:

Identification and mathematical properties of the exponential phase

Fluorophore background noise

Detection threshold (Cp/Ct/Cq)

4- PCR efficiency:

Definition and use E=10 -1/slope

Calculation of efficiency by linear regression of a dilution range

Primer validation: dynamics, linearity and detection limit

Standardization and validation of PCRQ manipulations (intra and inter-run)

The essential checks

5- Quantification by real-time PCR:

RNA and genomic DNA

Absolute quantization

Relative quantification - With and without efficiency correction (2ddCT)

Quantification strategy based on biological context

6- Standardization strategy:

Definition of reference genes (housekeeping gene)

Choice and validation of reference genes: arithmetic or geometric mean?

Free tools to know (Genorm, Bestkeeper, Rest)

7- Results:

Reproducibility

Sensitivity

Precision (exact/relative)

Technical and biological replicates

Presentation of results: Number of copies, comparison, fold change, fold increase

Some software to know or configure Excel?

8- Publication and PCRQ:

The MIQE (minimum information for publication of quantitative real-time PCR experiments) or the minimum required for publication

B- Quantitative PCR: practical aspect

This section aims to highlight the importance of the extreme rigor required in this technique at each stage if reproducibility and reliability of the results are desired.

The practical aspects presented follow the recommendations of the MIQE.

- Precautions and biological risks

- Setting up a quantification project: points to consider

- Equipment: thermocyclers, PCR circuit, Nanodrop, bio analyzer

- Consumables

- The kits

Protocols and optimization

Samples, RNA, cDNA, DNA: nature, quality, extraction, quantification, conservation

Reverse transcription: A key step to constantly optimize

RNA Quantity

Dnase treatment

The different enzymes

Priming strategy and detection sensitivity

(oligotDT random primers or specific primer)

RT Performance and Verification

RT controls

RT kits

cDNA

Detect PCR inhibitions

The primers

The calibration range

Contaminations and cross-contaminations

C- Different approaches to genotyping or polymorphism in PCRQ (By appointment)

Hybridization probes

Digital PCR

HRM (High Resolution Melt Analysis)

D- PCRQ primers designs (by appointment)

Primer quality is a key aspect of real-time PCR. This section aims to highlight its impact on results, make design accessible, and demonstrate that a good strategy can save a lot of time and improve efficiency.

Why it is (always) better to design the primers yourself:

Effective Design Strategy in 5 Easy Steps:

HUGO gene nomenclature and bio-info portal

TOGETHER: knowing your gene to define the sequence to be amplified

Intron exon organization; coding sequences, UTR, SNIP; isoforms

Design software and tools

- Beacon designer: why it's the best .BLAST and MFOLD

Parameters to specify for primers and amplicons

- Roche UPL

- Primer Blast

- Oligonucleotide banks

Amplify: highly predictive and free electronic PCR

Ordering, testing and validation of oligos

E- Analysis and exploitation of results (by appointment)

Programming and Run Analysis: Exploring the platform's instrument software

Calculation and exploitation in Excel.

Presentation of free tools for calculations or normalization: REST, Bestkeeper, Genorm.