qPCR in immune system monitoring :

a high throughput method

M. DOOMS, F.DEPEINT, A.M. ABDELNOUR, Institut Polytechnique LaSalle Beauvais (France)

E-mail: afif.adbelnour@lasalle-beauvais.fr

In clinical trials, time is often one of your worseopponent, among others including variability andmoney concerns. Besides these considerations,subjects/patients well being must be considered. Inclinical trials involving molecular biology, this workpackage is often one of the most time consumingand the most expensive.Based on these facts, we developed in our lab a newmethod focused on immune system based on thecombination of a inflammatory model with geneexpression monitoring. This method is MIQEcompliant.

At a glance protocol :

qPCR protocol: 100 ng of cDNA is used for each

reaction. The mix used is Solaris Master mix and

primers are contained with probes in Solaris

Predesigned assays. The mix is ready to use at a2x concentration. Its blue colour helps in

handling, since such small volumes are

sometimes hard to see if colourless. The

Predesigned assay contains primers and probes

designed by Solaris, which is also convenient,

since it dramatically reduces the risk of mismatchwhile pipeting.

Solaris predesigned assay: The pre designed assay helps a lot in performing analysis.

~ No time required for designing primers and probes

~ All in the same tube: very convenient, reduces pipeting concerns

Superbases and MGB: The amplicons are highly optimized, thanks to reduced size of primers and

probes.

~ Incorporation of superbasesTM, wich increases the melting temperature for smaller primers

and minor groove binder (MGB)

~ Increases stability of the probes

~ Smaller primers and probes allows wider coverage of the genome, so over 98% of the

genome is covered

~ Fluorescence relies on structural change rather than hydrolysis

RESULTS: As expected, LPS stimulationinduces an inflammatory response. This

is show by increase in immune markers.

Due to low number of samples (only

three), the statistical power is not very

high, but thanks to MIQE, the biological

relevance is clear.

About us: Afif AbdelNour is a very

experienced researcher in

nutrigenomics at LaSalle Beauvais.

Maxime Dooms is a 5th year

student (MsC) at the same

institute, undergoing an internship

focused on molecular biology.

Conflict of interest: this poster is

supported by Thermo Fisher

Scientific.

LPS

stim

ula

tio

n Blood Sampling(500 µL)

LPS (50ng)

Incubation at37°C

H+6 RNA extraction

Qualityassesment

RT and Qualityassesment

H+2

4 RNA extraction

Qualityassesment

RT and qualityassesment

geNorm is a plug in for Microsoft

Excel. It helps in choosing the right

reference genes, based on qPCR

results.

RNA integrity control has been

validated on Agilent RNA 6000

nano kit.

In this experiment, the control group is blood

from healthy subjects without in vitro LPS

stimulation. The test group’s blood have

received LPS and has been incubated at37°C for different times.

Samples have not been thaw more than

once and always on ice. Storagetemperature was -80°C.

DNAse treatment is done after extraction,

right before RT.

cDNA synthesis is done using QuantiTect RT

kit from QIAGEN, without modification (100

ng). Purity is assessed with OD 260,280 and

130. The ratios 260/280 are between 1,76

and 1,84, 260/230 between 2,11 and 2,29.

cDNA are stored on ice before qPCR (a few

hours).

References; Bustin et al, The MIQE guidelines: Minimum information for publication of quantitative

real-time PCR experiments, 2009: Pouillart et al, NUTRIOSE®, a prebiotic low-digestible

carbohydrate stimulates gut mucosal immunity and prevents TNBS- induced colitis in piglets, 2009

Efficience curves have been done

for every gene. Results have been

normalized according to these

values.

Relative expression of three immune markers