After organ retrieval, tissue was immediately fixed in RNAlater. Total mRNA was extracted using the RNeasy mini kit system (Qiagen, Hilden, Germany) and transcribed with Quiagen mini kits. For quantitative PCR (qPCR), 1 μg of DNase-treated total RNA was reverse transcribed using Superscript II Reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and qPCR was performed on a Lightcycler 420 II (Roche Diagnostics, Penzberg, Germany) using FastStart Sybr-Green. Gene-specific primers for CXCL13 (Primer-sequence: fwd-TCT GGA CCA AGA rev-TGA AGA AAG TT) and monocyte chemoattractant protein-1 (MCP-1; Mm_Ccl2_1_SG QuantiTect Primer Assay QT00167832) were used. Quantification was carried out using QGene software.
Lightcycler 420 2
Manufactured by Roche
Sourced in Germany
The Lightcycler 420 II is a real-time PCR instrument designed for quantitative nucleic acid analysis. It features a compact, modular design and supports a variety of sample formats. The Lightcycler 420 II enables precise and reproducible detection and quantification of DNA and RNA targets.
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Lab products found in correlation
3 protocols using lightcycler 420 2
1
Quantification of CXCL13 and MCP-1 mRNA
2
Quantitative PCR for Inflammatory Genes
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Tissue sections were stored in RNA-later immediately after organ retrieval. Total RNA was extracted using the RNeasy mini kit system (Qiagen, Hilden, Germany) and transcribed using Superscript II Reverse transcriptase (Invitrogen). Quantitative (q) PCR was performed on Lightcycler 420 II (Roche Diagnostics, Penzberg, Germany) using FastStart Sybr-Green chemistry. Gene-specific primers for IL–6 (Quantitec QT00098875, Qiagen) and MCP–1 (Quantitec QT00167832, Qiagen) were used for the gene of interest and HPRT served as house keeping gene for normalization (Quantitec QT00166768, Qigaen). Quantification was carried out using qgene software.
3
Quantitative PCR Analysis of Renal mRNA
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For mRNA work-up one part of the kidneys was fixed in RNAlater immediately. For total RNA extraction the RNeasy mini kit system (Qiagen, Hilden, Germany) was used and RNA was transcribed with Qiagen mini kits. For quantitative PCR (qPCR) 1 µg of DNase-treated total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and qPCR was performed on a Lightcycler 420 II (Roche Diagnostics, Penzberg, Germany) using FastStart Sybr-Green chemistry. Gene-specific primers for MCP-1 (QT00167832) and IL-6 (QT00098875) were used. For normalization HPRT (QT00166768) was used as housekeeping gene. Quantification was carried out using qgene software.
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