Quantitative Real-Time PCR Analysis of Gene Expression

Mouse and human brain tissue samples and cultured primary mouse astrocytes were homogenized using a Precellys homogenizer with 1.4-mm ceramic beads (5,000 rpm, 30 s, Peqlab). RNA was isolated using the NucleoSpin RNA II kit (Macherey–Nagel) according to the manufacturer’s instructions. RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Peqlab). RNA (0.1 µg) was reverse transcribed to cDNA using random primers (Roche) and Superscript III (Life Technologies). Quantitative real-time PCR was performed in a Rotorgene3000 (Corbett Research) using the Rotor-Gene SYBR Green PCR kit and the Quantitect primer assay HsQPCTL (Qiagen), or specific primers for HsCCL2, MsCCL2 and MsQPCTL synthesized by Metabion (Martinsried, Germany) [8 (link), 23 (link)]. Relative amounts of gene expression were determined with the Rotorgene software version 6.1 in comparative quantitation mode. Normalization was done against the most stably expressed reference gene YWHAZ identified using Normfinder [3 (link)]. The PCR was verified by product melting curves and single amplicons were confirmed by agarose gel electrophoresis.

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Hartlage-Rübsamen M., Waniek A., Meißner J., Morawski M., Schilling S., Jäger C., Kleinschmidt M., Cynis H., Kehlen A., Arendt T., Demuth H.U, & Roßner S. (2015). Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer’s disease. Acta Neuropathologica, 129(4), 565-583.

Publication 2015

NucleoSpin RNA II kit NanoDrop 2000 spectrophotometer Superscript III Rotorgene3000 Rotor-Gene SYBR Green PCR kit

Agarose gel electrophoresis Assay Astrocytes Brain Cdna Gene Gene expression Human Mouse Primers Quantitative real time pcr Rna ii Sybr green Tissue

Corresponding Organization :

Other organizations : Leipzig University, Fraunhofer Society, Martin Luther University Halle-Wittenberg

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Variable analysis

independent variables

Mouse and human brain tissue samples
Cultured primary mouse astrocytes

dependent variables

Gene expression of CCL2 in human and mouse samples
Gene expression of QPCTL in human and mouse samples

control variables

RNA isolation using NucleoSpin RNA II kit
RNA concentration measurement using NanoDrop 2000 spectrophotometer
Reverse transcription to cDNA using random primers and Superscript III
Quantitative real-time PCR using Rotor-Gene SYBR Green PCR kit
Normalization against the reference gene YWHAZ

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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