Régulation des populations de Nématodes gastro-intestinaux ...

Résultats
RNA were digested with 1U of RNase-free DNAse (Promega) for 1h at 37°C as to remove
any trace of genomic DNA, and then incubated 10 min at 65°C with DNase Stop Solution.
Treated RNA was used for single-stranded cDNA synthesis. They were incubated 10 min at
65°C with Random Primers oligonucleotides (Invitrogen, reference 48190-011) and chilled on
ice; a mix containing M-MLV transcriptase (400 IU/sample, Invitrogen, reference 28025-
013), 5x first-strand buffer (Life Technologies), dithiothreitol (DTT) 0.1 M (Life
Technologies), 40 U of RNase Out Ribonuclease Inhibitor (Invitrogen, reference 10777-019),
10 mM of each four dNTPs (Promega) was then added and the mixture incubated for 1h at
42°C. Reaction was heat-inactivated at 95°C for 5 min and kept at –20°C until used. For
quantitative PCR, cDNA were used at dilutions of 1:100.
2.3.2. PCR primers
Specific primers are listed in Table 2. For each target cDNA, a primer pair was
determined using Primer Express Software (Applied Biosystems) for SYBR Green real-time
PCR assay on a GeneAmp 5700 Sequence Detection System (Applied Biosystems) and based
on known ovine gene sequences (β-actin, IFN-γ, TNF-α, IL-3, IL-4, IL-5, IL-10, IL-12p40).
In cases where the ovine gene sequence was not known (IL-13), a consensus sequence was
created, based on a minimum of three known sequences in other mammalian species. A first
primer pair was then designed using Primer 3 Software as to amplify the largest possible
mRNA in all aligned sequence. PCR amplification on reverse transcript RNA obtained from
ovine peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A (ConA, 10
µg/mL, 24 hours in a 5% CO2 and 37°C atmosphere) was then performed. Amplicons were
purified before cloning in TopoCloning system (Invitrogen) and sequenced (using M13
universal primers). The sequences thus obtained were then compared with GenBank database
as to validate the nature of the amplified product. After validation a new set of primers
suitable for quantitative PCR was designed using the obtained sequence.
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