Gene Expression

Perform gene expression on tissue samples of the ventral midbrain (containing the substantia nigra pars compacta, SNpc).

At due time points, homogenize tissue samples in 1mL of QIAzol Lysis Reagent (Qiagen, #79306) using a rotor-stator homogenizer.

Isolate the total RNA from homogenized tissue samples using RNeasy Lipid Tissue Kit (Qiagen, #74804) including Dnase digestion.

At the end, re-dissolve RNA samples in 30µL of RNase-free water and determine their concentrations spectrophotometrically by A₂₆₀ (Nanodrop-ND 1000).

Perform cDNA synthesis using Retroscript Kit (Ambion #1710, Austin, Texas) according the manufacturer’s instructions.

Add 50µL of water solution containing 0.5µg of each pool to an equal volume of 2X TaqMan Universal PCR Mastrer Mix (Applied Biosystems).

Perform real-time quantitative PCR using Taqman^TMAssay Reagents on an Step One Detection System (Applied Biosystems) according to manufactures protocol.

Process tissue samples as above.

Lyse the cell samples in lysis buffer (Qiagen) and store at -80°C until the RNA extraction following manufacture’s instructions.

Remove residual genomic DNA by incubating with DNase I, RNase-free (Qiagen) and elute from the RNeasy mini columns with RNase-free water.

Quantify the amount of total RNA was using a NanoDrop ND-100 (Nano Drop Technologies) and synthesize the cDNA from 2µg of total RNA using the Retroscript Kit (Ambion).

After purification using QIAquick PCR Purification kit (Qiagen), use 250ng of cDNA for Real-time PCR using pre-developed Taqman Assay Reagents (Applied Biosystems).

Perform real-time quantitative PCR with Step One Detection System (Applied Biosystems) according to manufacturer protocol.

Using the delta delta C_t (2^-ΔΔCt) comparative method, quantification of the abundance of target gene expression was determined relative to β-actin with respect to the control group, express the results as arbitrary units (AU).

Indicate relative fold changes over WT in the respective treatment groups.