Rotor gene q 2plex cycler

RNA was isolated using a NucleoSpin^® RNA/Protein kit (Macherey-Nagel, Düren, Germany). Sample quality and purity were ensured by randomly testing using QIAxcel^® RNA analyses (Qiagen, Hilden, Germany). Purified mRNA was transcribed into cDNA with a QuantiTect^®Reverse Transcription Kit (Qiagen, #205313). Transcripts of complement components, receptors, and inflammation-associated markers were analyzed using a Rotor-Gene SYBR^®Green PCR Kit either with QuantiTect Primer Assays (Qiagen, Supplementary Table 1) or in-house-designed primer pairs (Metabion, Planegg, Germany, Supplementary Table 1) in a Rotor Gene Q 2plex cycler (Qiagen), using the following conditions: hold (95°C, 5 min), cycling (95°C, 5 sec; 60°C, 10 sec; 72°C, 20 sec; 40 cycles). Data were normalized to GAPDH housekeeper expression, analysed by using the 2^-ΔΔCT method. Values were depicted on a linear scale using log₂-transformed scores to equally visualize increases and decreases in expression levels.

mRNA was isolated using the NucleoSpin^® RNA kit (Macherey-Nagel, Düren, Germany, #740955) and transcribed into cDNA with the QuantiTect^® Reverse Transcription Kit (Qiagen, Hilden, Germany, #205313). Transcripts of complement components, regulators, receptors and inflammation-associated markers were analyzed (i) by PCR (as described in Section 2.2.) and subsequent 2% agarosegel separation for cDNA fragment visualization, or (ii) by qPCR using Rotor-Gene SYBR^® Green PCR Kit (Qiagen) in a Rotor Gene Q 2plex cycler (Qiagen) either with QuantiTect Primer Assays (Qiagen, Table S2), or in-house designed primers for IL1B, VIM and SMA1 (Metabion, Planedd, Germany, Table S1). Data were normalized to the housekeeper GAPDH expression and fold change was calculates using 2(-Delta Delta C(T)) method [40 (link)]. Data are visualized on a linear scale using log2-transformed scores [41 (link)].