Services

Quantitative PCR Plateform

Responsable : Eric Jacquet (01 69 82 46 24)
Ingénieur Plateforme : Naima.Nhiri

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Sample preparation

 

Grinding and extraction

For the preparation of samples from tissues or organs, it will be necessary to perform grinding. This grinding will be carried out on ceramic beads using the Precellys 24 with Cryolys refrigeration in single-use tubes. If necessary, the extraction can be done in Trizol to improve yields.

RNA purification

RNA purification will be carried out on a column using a Macherey-Nagel or Qiagen kit. Purification can be automated on a Qiacube instrument depending on the project. Extractions made by grinding in Trizol will be systematically repurified on a column with the kits.

Dosage and quality control

The concentration of the purified samples will be measured with Nanodrop or Qubit. RNA quality control will be performed on an Agilent 2100 Bioanalyzer chip. The measurement of the integrity of the purified RNAs is necessary before using the samples for the reactions of quantitative PCR. The service includes the preparation of RNA 6000 Nano or RNA 6000 Pico chips, with samples loading in simplicat and certain samples in duplicate. Analysis of profiles and verification of quality indices of RNA preparations (RIN).

Reverse transcription

The reverse transcription step allows the preparation of cDNAs from purified RNA samples. The choice of the protocol and the reverse transcription kit will be made according to the needs of the project.

 

Quantitative PCR services

 

Design and validation of oligonucleotide pairs

For quantitative PCR projects in SybrGreen, our team offers to design your primers and order them for you. Services include primers optimization and QPCR reaction efficiency measurement. A minimum of 1 pair of oligonucleotides is designated for each target gene and each reference gene. Some genes will require the design of several pairs to select the best in specificity and efficiency.

Selection and design of TaqMan probes

Quantitative PCR projects with TaqMan probes can be developed using inventoried or custom-ordered TaqMan probes. The TaqMan probes of the reference genes are available on the platform.

QPCR Sybr Green and TaqMan in microplate (medium-throughput)

Projects developed in SybrGreen will be produced in 384 microplate on the QuantStudio 12K. For each project, internal efficiency curves of the pairs of primers and negative controls for non-contamination will be added to the plan of the experiment.

QPCR in TaqMan Array Card (high-throughput in microvolume)

The platform has a robotic 7900HT allowing the realization of projects in high-throughput TaqMan probes using microfluidic cards (TaqMan Array Card). The use of these cards makes it possible to quickly determine the expression profile of a large number of genes on several samples. The cards are preloaded with a TaqMan probe allowing the simultaneous measurement of the expression of 16 to 384 genes (reference genes and genes of interest). This highly sensitive, reproducible and automated technique allows up to 5000 assays to be performed per day using volumes of 1 µL per reaction. Support for the design of the TaqMan Array Card is provided by the platform team. The design and ordering of TaqMan Array Cards can also be supported by the platform.

QPCR in Open Array Plate (very high throughput in nanovolume)

For the requirements of very high throughput transcriptome study projects, the platform is equipped with a new generation QPCR device. The platform has a QuantStudio 12K Flex equipped with an Open Array block and its AccuFill loading robot. This device can process up to 12,000 QPCRs per 2 hour run in a reaction volume of 10 to 33nL. The performance of this type of equipment is suitable for screening projects requiring the analysis of several hundred samples or target genes.

Analysis, standardization and data processing

Data from quantitative PCR projects will be analyzed by platform staff. These analyzes include: quality of amplification curves, export of raw values, analysis of Ct values ​​and technical replicates, calculation of group means, search and selection of the best reference genes by GeNorm and NormFinder software, calculation of variations of expression between samples.