Quantitative RT-PCR for Gene Expression

Total RNA was isolated using the NucleoSpin RNA mini kit (740955, Macherey-Nagel, Düren, Germany), cDNA synthetized with iScript cDNA Synthesis Kit (1708891BUN, BioRad, Feldkirchen, Germany) and RT-qPCR performed in technical triplicates with ABsolute SYBR Green Mix (AB-1158B, Thermo Fisher Scientific, Darmstadt, Germany) according to the instructions of the manufacturers using Stratagene Mx3000P qPCR System (Agilent Technologies, Waldbronn, Germany). Results were evaluated by the Cy0 method 58 (link) with RPL27 used for normalization. The following primers were used:
RPL27: AAAGCTGTCATCGTGAAGAAC and GCTGTCACTTTGCGGGGGTAG;
IL6: AGGAACAAGCCAGAGCTGTGCAGATG and TTTGTGGTTGGGTCAGGGGTGGTTA; THBS1: TCTCTGACCTGAAATACGAATGTAG and AAGGAAGCCAAGGAGAAGTG; CCL20: GCTGCTTTGATGTCAGTGCT and GCAGTCAAAGTTGCTTGCTTC;
LPAR1: ATTTCACAGCCCCAGTTCACA and ACCAGCTTGCTGACTGTGTT;
LPAR2: GCCTGGTCAAGACTGTTGTCA and CCAGGACATTGCAGGACTCA;
LPAR3: CCAACGTCTTGTCTCCGCATA and CCGGGGTCCAGCATACCA

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Ojasalu K., Lieber S., Sokol A.M., Nist A., Stiewe T., Bullwinkel I., Finkernagel F., Reinartz S., Müller-Brüsselbach S., Grosse R., Graumann J, & Müller R. (2023). The lysophosphatidic acid-regulated signal transduction network in ovarian cancer cells and its role in actomyosin dynamics, cell migration and entosis. Theranostics, 13(6), 1921-1948.

Publication 2023

NucleoSpin RNA mini kit iScript cDNA Synthesis Kit ABsolute SYBR Green Mix Stratagene Mx3000P qPCR System

Ccl20 Cdna Primers Sybr green Synthesis Thbs1

Corresponding Organization : Philipps University of Marburg

Other organizations : Max Planck Institute for Heart and Lung Research, University of Freiburg

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Variable analysis

independent variables

RNA isolation method (NucleoSpin RNA mini kit)
CDNA synthesis method (iScript cDNA Synthesis Kit)
RT-qPCR method (ABsolute SYBR Green Mix)

dependent variables

Expression levels of genes: IL6, THBS1, CCL20, LPAR1, LPAR2, LPAR3

control variables

Normalization gene (RPL27)
Thermal cycler (Stratagene Mx3000P qPCR System)

controls

No positive or negative controls were explicitly mentioned.

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