|Application:||Gene expression microarray analysis|
|Number of samples:||365|
|Release date:||Oct 26 2014|
|Last update date:||Aug 13 2018|
|Diseases:||Metabolism, Inborn Errors, Androgen-Insensitivity Syndrome|
|Dataset link||Genome-wide profiling of whole blood from patients with defects in Toll-like receptors (TLRs) and IL-1Rs (the TIR pathway) signaling|
A total of 22 blood samples were collected from three groups of subjects: 4 patients with complete MYD88 deficiency, 4 patients with complete IRAK4 deficiency, and 14 healthy donors. Mutations from IRAK4 and MYD88 patients are loss-of-function. The subjects were recruited in two different batches: first set includes 4 healthy controls, MYD88 P1 and P2, IRAK4 P1 and P2 patients. Second set includes 10 different healthy controls, MYD88 P3 and P4 and IRAK4 P3 and P4. Healthy individuals are considered so based on past medical history (no recurrent infections). They were also healthy at the time of sampling. From each subject, peripheral blood was drawn into sodium heparin vacuum tubes on clinic site (Necker Hospital, Paris). Right away, 500Âµl whole blood was diluted with the same amount of RPMI before adding different stimulus. Diluted whole blood was activated with a wide range of ligands (16 conditions total, see below) in a volume of 50 Âµl, for 2 hours along with 10Âµg/ml of polymyxin B to clear LPS contamination in reagents. Whole blood was lysated with Tempus solution (from Tempus tubes, Applied Biosystems) to stabilize the RNA at 1:3 ratio after restimulation. The lysates were kept at -80ÂºC temperature until mRNA extraction. A total of 365 samples were analyzed. Choice of agonists: PAM3 (TLR1/2) and PAM2 (TLR2/6), 100 ng/ml final concentration, InvivogenÂ®; poly(I:C) 25 Âµg/ml final concentration, InvivogenÂ® (TLR3); LPS 100 ng/ml final concentration, SigmaÂ® (TLR4); Flagellin 100 ng/ml final concentration, InvivogenÂ® (TLR5); 3M13 (TLR7) and 3M2 (TLR8), 3Âµg/ml final concentration, 3M pharmaceuticalsÂ®; R848 3Âµg/ml final concentration, InvivogenÂ® (TLR7/8); CpG-D19 and CPG C (TLR9), 3Âµg/ml final concentration, from collaborator. Agonists for IL-1R: IL-1b 20ng/ml final concentration, IL18 and IL33 50ng/ml final concentration, from RaD systemsÂ®; agonist for TNFa receptor, TNFa, 20ng/ml final concentration, from RaD systemsÂ®; PMA 25 ng/ml + Ionomycin 1Âµg/ml final concentration, SigmaÂ®; heat killed whole bacteria: 3 pneumococcal strains (R6, 5.106 partical/ml final concentration; R8450 108 partical/ml final concentration; R11470, 5.106 partical/ml final concentration, heat killed at 65Â°C for 15 min) from collaborator, and Staphylococcus aureus (SAC), 107 partical/ml final concentration, from InvivogenÂ®. No stimulation serves as negative control and PMA/ionomycin stimulation as positive control.
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