Interferon Signature Quantification Protocol

Total RNA was extracted from whole blood using a PAXgene (PreAnalytix) RNA isolation kit. RNA concentration was assessed using a spectrophotometer (FLUOstar Omega, Labtech). Quantitative reverse transcription PCR analysis was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems), and cDNA derived from 40ng total RNA. The relative abundance of target transcripts, measured using TaqMan probes for IFI27 (Hs01086370_m1), IFI44L (Hs00199115_m1), IFIT1 (Hs00356631_g1), ISG15 (Hs00192713_m1), RSAD2 (Hs01057264_m1) and SIGLEC1 (Hs00988063_m1) was normalized to the expression level of HPRT1 (Hs03929096_g1) and 18s (Hs999999001_s1) and assessed with the Applied Biosystems StepOne Software v2.1 and DataAssist Software v.3.01. For each of the six probes, individual (patient and control) data were expressed relative to a single calibrator (control C25). As previously described, the median fold change of the six ISGs, when compared to the median of the combined 29 healthy controls, was used to create an interferon score for each patient^{9 (link)}. RQ (relative quantification) is equal to 2^−ΔΔCt i.e. the normalized fold change relative to a control. When a patient was assayed on more than one occasion, the data for repeat measurements were combined to calculate a mean value (using Applied Biosystems DataAssist Software v.3.01).

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Rice G.I., del Toro Duany Y., Jenkinson E.M., Forte G.M., Anderson B.H., Ariaudo G., Bader-Meunier B., Baildam E.M., Battini R., Beresford M.W., Casarano M., Chouchane M., Cimaz R., Collins A.E., Cordeiro N.J., Dale R.C., Davidson J.E., De Waele L., Desguerre I., Faivre L., Fazzi E., Isidor B., Lagae L., Latchman A.R., Lebon P., Li C., Livingston J.H., Lourenço C.M., Mancardi M.M., Masurel-Paulet A., McInnes I.B., Menezes M.P., Mignot C., O’Sullivan J., Orcesi S., Picco P.P., Riva E., Robinson R.A., Rodriguez D., Salvatici E., Scott C., Szybowska M., Tolmie J.L., Vanderver A., Vanhulle C., Vieira J.P., Webb K., Whitney R.N., Williams S.G., Wolfe L.A., Zuberi S.M., Hur S, & Crow Y.J. (2014). Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling. Nature genetics, 46(5), 503-509.

Publication 2014

FLUOstar Omega TaqMan Universal PCR Master Mix StepOne Software v2.1 DataAssist Software v.3.01

Blood Cdna Interferon Isolation Patient Reverse transcription

Corresponding Organization :

Other organizations : Manchester Academic Health Science Centre, University of Manchester, Harvard University, Fondazione Istituto Neurologico Nazionale Casimiro Mondino, Institut des Maladies Génétiques Imagine, Assistance Publique – Hôpitaux de Paris, Inserm, National Health Service, Alder Hey Children's NHS Foundation Trust, Istituti di Ricovero e Cura a Carattere Scientifico, Fondazione Stella Maris, Centre Hospitalier Universitaire Dijon Bourgogne, University of Florence, Meyer Children's Hospital, University of Colorado Denver, NHS Ayrshire and Arran, University of Sydney, Children's Hospital at Westmead, Royal Hospital for Children, KU Leuven, Université de Bourgogne, Hôpital d'Enfants, University of Brescia, Centre Hospitalier Universitaire de Nantes, Génétique Médicale & Génomique Fonctionelle, McMaster University, McMaster Children's Hospital, Université Paris Cité, Délégation Paris 5, Leeds Teaching Hospitals NHS Trust, Universidade de São Paulo, Clinics Hospital of Ribeirão Preto, Istituto Giannina Gaslini, University of Glasgow, Pitié-Salpêtrière Hospital, Sorbonne Université, University of Milan, Ospedale San Paolo, University College London, Great Ormond Street Hospital, Hôpitaux Universitaires Paris-Ouest, University of Cape Town, Red Cross War Memorial Children's Hospital, Southern General Hospital, Children's National, Université de Rouen Normandie, Hôpital Charles-Nicolle, Centro Hospitalar de Lisboa Central, Hospital de Dona Estefânia, National Institutes of Health, Office of the Director, Boston Children's Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Relative abundance of target transcripts for IFI27, IFI44L, IFIT1, ISG15, RSAD2, and SIGLEC1

control variables

Total RNA extracted from whole blood using a PAXgene (PreAnalytix) RNA isolation kit
RNA concentration assessed using a spectrophotometer (FLUOstar Omega, Labtech)
CDNA derived from 40ng total RNA
Quantitative reverse transcription PCR analysis performed using the TaqMan Universal PCR Master Mix (Applied Biosystems)
Relative abundance of target transcripts measured using TaqMan probes
Normalized to the expression level of HPRT1 and 18s
Assessed with the Applied Biosystems StepOne Software v2.1 and DataAssist Software v.3.01
Patient and control data expressed relative to a single calibrator (control C25)
Median fold change of the six ISGs compared to the median of the combined 29 healthy controls used to create an interferon score for each patient

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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