Real-Time PCR Analysis of Mouse and Human Immune Markers

For mouse skin, total RNA was isolated as stated above. For human PBMCs, total RNA was isolated from PBMC pellets with a Trizol extraction system (TriFast, PEQLAB GmbH, Erlangen, Germany), cDNA synthesis and quantitative RT-PCR was performed as previously described^{69 (link),70 (link)}. The Applied Biosystems StepOne Real-Time PCR system (Applied Biosystems, Forster City, CA) and TaqMan® Fast Universal PCR Master Mix for TaqMan assays (Applied Biosystems) were used for analysis. β-actin and cyclophilin were used as internal housekeeping controls and the quantitative analysis was performed with the ΔΔCT method. The following TaqMan® Gene Expression Assays (Applied Biosystems) were used: mouse Actb (Mm00607939_s1), mouse Tnf (Mm00443258_m1), human TNF (Hs99999043_m1), human PPIA (Hs99999904_m1).
All pre-clinical experimental analyses were performed in a blinded manner.

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Mirastschijski U., Schwab I., Coger V., Zier U., Rianna C., He W., Maedler K., Kelm S., Radtke A., Belge G., Lindner P., Stahl F., Scharpenberg M., Lasota L, & Timm J. (2020). Lung Surfactant Accelerates Skin Wound Healing: A Translational Study with a Randomized Clinical Phase I Study. Scientific Reports, 10, 2581.

Publication 2020

TriFast StepOne Real-Time PCR system TaqMan® Fast Universal PCR Master Mix for TaqMan assays TaqMan® Gene Expression Assays

Actin Assays Cdna Cyclophilin Gene expression Human Mouse Pellets Rt pcr Skin Synthesis Trizol

Corresponding Organization : University of Bremen

Other organizations : Klinikum Bremen-Mitte, Medizinische Hochschule Hannover, Leibniz University Hannover

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Variable analysis

independent variables

Mouse skin vs. human PBMCs

dependent variables

Expression of Tnf in mouse skin
Expression of TNF in human PBMCs

control variables

Total RNA isolation method (Trizol extraction for human PBMCs)
CDNA synthesis and quantitative RT-PCR procedure (as previously described in references 69 and 70)
Housekeeping genes (β-actin and cyclophilin) used for normalization
Quantitative analysis method (ΔΔCT method)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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