Computational protocol: Heat shock protein 60: an endogenous inducer of dopaminergic cell death in Parkinson disease

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Protocol publication

[…] The Hsp60 and TH primers for quantitative PCR were designed by primer express 3.0 (Applied Biosystems, Darmstadt, Germany) and their specificity was confirmed by blastn analysis. Their sequences were as followed: Hsp60_fwd: 5′-GCC GCC CCG CAG AA-3′, Hsp60_rev: 5′-CCT GGA CAC CGG TCT CAT CT-3′, TH_fwd: 5′-GCA CCT TCG CGC AGT TCT-3′, TH_rev: 5′-ACA GCG TGG ACA GCT TCT CA-3′. Mesencephalon ss PD patients and age-matched control subjects (n = 5 per group) were provided by the INSERM UMR_S1127 brain bank. The SN was dissected from frozen slides. Total RNA was extracted with Trizol (Invitrogen, Cergy Pontoise Cedex, France) and the quantity and quality were determined by Nanodrop and Agilent, respectively. One microgram of total RNA was reverse-transcribed with Superscript III RT-kit (Invitrogen, Cergy Pontoise Cedex, France) according to the manufacturer’s instructions. qPCR was performed with SYBR GreenER TM qPCR SuperMix kit (Invitrogen, Cergy Pontoise Cedex, France) in ABI 7500 real time thermal cycler (Applied Biosystems, Darmstadt, Germany). The relative mRNA expression levels were normalized by the geometric mean of two housekeeping genes (GAPDH and HPRT) with qBaseplus software (Primerdesign, Southampton, UK). […]

Pipeline specifications

Software tools Primer Express, BLASTN
Application qPCR
Organisms Mus musculus, Homo sapiens
Diseases Parkinson Disease
Chemicals 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-Methyl-4-phenylpyridinium