Quantitative analysis of midbrain gene expression

Midbrain from mice aged 6 months were extracted with Trizol (Invitrogen) following manufacturer’s instructions. Before cDNA synthesis, 1 μg of RNA was digested with DNase I Amplification Grade (Invitrogen). Reverse transcription was performed with SuperScript III Reverse Transcriptase (Invitrogen). Subsequently, expression levels were measured with the StepOnePlus Real-Time PCR System (Applied Biosystems) using cDNA from 25 ng RNA, 10 μl of FastStart Universal Probe Master (Rox) Mix (04914058001, Roche) and 1 μl of one of the following TaqMan Assays (Applied Biosystems): Adora2b (Mm00839292_m1), Dapk1 (Mm01352536_m1), Dcaf17 (Mm01334341_m1), Dctn5 (Mm00727515_s1), Homer1 (Mm00516275_m1), Mrpl9 (Mm00659648_m1), Mrpl9 (Mm00659648_m1), Pink1 (Mm00550827_m1) as independent control of genotypes, Rab42 (Mm01187370_g1), Tmem181a (Mm02581460_g1), Xaf1 (Mm01248390_m1), Lect1 (Mm00495291_m1) and Tbp (Mm00446973_m1) as endogenous control. Expression changes were analyzed both with the 2^−ΔΔCt method, as previously described (77 (link)).

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Gispert S., Brehm N., Weil J., Seidel K., Rüb U., Kern B., Walter M., Roeper J, & Auburger G. (2014). Potentiation of neurotoxicity in double-mutant mice with Pink1 ablation and A53T-SNCA overexpression. Human Molecular Genetics, 24(4), 1061-1076.

Publication 2014

Trizol DNase I Amplification Grade SuperScript III Reverse Transcriptase StepOnePlus Real-Time PCR System FastStart Universal Probe Master (Rox) Mix TaqMan Assays

Adora2b Assays Cdna Dnase i Genotypes Mice Midbrain Reverse transcriptase Reverse transcription Synthesis Trizol Xaf1

Corresponding Organization :

Other organizations : Goethe University Frankfurt, University of Tübingen

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Variable analysis

independent variables

Genotype

dependent variables

Expression levels of Adora2b, Dapk1, Dcaf17, Dctn5, Homer1, Mrpl9, Pink1, Rab42, Tmem181a, Xaf1, Lect1

control variables

Midbrain from mice aged 6 months
RNA extraction using Trizol (Invitrogen)
DNase I Amplification Grade (Invitrogen) treatment of 1 μg of RNA
Reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen)
Measurement of expression levels using StepOnePlus Real-Time PCR System (Applied Biosystems)
CDNA from 25 ng RNA
FastStart Universal Probe Master (Rox) Mix (04914058001, Roche)
TaqMan Assays (Applied Biosystems)

positive controls

Tbp as endogenous control

negative controls

Pink1 as independent control of genotypes

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