qPCR in immune system monitoring :
a high throughput method
M. DOOMS, F.DEPEINT, A.M. ABDELNOUR, Institut Polytechnique LaSalle Beauvais (France)
E-mail: [email protected]
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
