Real-Time qPCR Analysis of sFlt1, MX-1 and GAPDH

RNA was isolated using RNeasy kit (QIAGEN) and 0.5 μg of RNA per condition was used for the cDNA synthesis using iScript Reverse Transcription Supermix (BIO-RAD), and an ABI Veriti Thermocycler (Life Technologies). SsoAdvance Universal SYBR Green Supermix and CFX96 Real Time System from BIO-RAD were used following manufacturer’s instructions for volumes and reaction settings. Primer sequences for sFlt1, MX-1 and GAPDH genes were previously described (28 (link), 33 (link), 34 (link)), and purchased from IDT Integrated DNA Technologies.

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Andrade D., Kim M., Blanco L.P., Karumanchi S.A., Koo G.C., Redecha P., Kirou K., Alvarez A.M., Mulla M.J., Crow M.K., Abrahams V.M., Kaplan M.J, & Salmon J.E. (2015). Interferon-α and angiogenic dysregulation in pregnant lupus patients destined for preeclampsia. Arthritis & rheumatology (Hoboken, N.J.), 67(4), 977-987.

Publication 2015

RNeasy kit iScript Reverse Transcription Supermix ABI Veriti Thermocycler SsoAdvance Universal SYBR Green Supermix CFX96 Real Time System

Cdna Gapdh Genes Primer Reverse transcription Sybr green Synthesis

Corresponding Organization :

Other organizations : Hospital for Special Surgery, Cornell University, Albert Einstein College of Medicine, Yeshiva University, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Beth Israel Deaconess Medical Center, Yale University

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Variable analysis

independent variables

RNA isolation using RNeasy kit (QIAGEN)
CDNA synthesis using iScript Reverse Transcription Supermix (BIO-RAD)
Primer sequences for sFlt1, MX-1 and GAPDH genes

dependent variables

Gene expression levels of sFlt1, MX-1 and GAPDH

control variables

0.5 μg of RNA per condition used for cDNA synthesis
SsoAdvance Universal SYBR Green Supermix and CFX96 Real Time System from BIO-RAD used for qPCR following manufacturer's instructions

controls

No positive or negative controls were explicitly mentioned

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