this protocol describes a method for quantifying gene expression in tissue samples, specifically in the ventral midbrain containing the substantia nigra pars compacta (snpc). the protocol involves RNA extraction, reverse transcription, and real-time quantitative PCR (qrt-pcr) using taqman assays. the process begins with homogenizing tissue samples in qiazol lysis reagent, followed by isolation of total rna using the rneasy lipid tissue mini kit, including dnase digestion. the rna is then re-solubilized in rnase-free water and quantified using a nanodrop. reverse transcription is performed using the retroscript kit, and cDNA is synthesized. the cDNA is then purified and used for qrt-pcr using taqman assay reagents on a step one detection system. the protocol uses β-actin as a housekeeping gene for normalization and embryonic mouse brain as a calibrator. gene expression levels are calculated using the delta delta ct (2^-ΔΔct) method, with results expressed as arbitrary units (au). the relative fold changes compared to wild-type (wt) are indicated for each treatment group. the protocol is open access and distributed under the creative commons attribution license. the authors are michela deleidi, bianca marchetti, federico bertoli, and carmela giachino from various institutions in germany and italy. the protocol was created on march 31, 2023, and is currently in a working status. the protocol is funded by the ASAP (aligning science across parkinson's) grant. the materials include qiazol lysis reagent, rneasy lipid tissue mini kit, nanodrop, retroscript kit, taqman universal pcr master mix, step one detection system, and other reagents. the protocol is intended for use in research on neurodegenerative diseases, particularly those involving mitochondria and inflammation.this protocol describes a method for quantifying gene expression in tissue samples, specifically in the ventral midbrain containing the substantia nigra pars compacta (snpc). the protocol involves RNA extraction, reverse transcription, and real-time quantitative PCR (qrt-pcr) using taqman assays. the process begins with homogenizing tissue samples in qiazol lysis reagent, followed by isolation of total rna using the rneasy lipid tissue mini kit, including dnase digestion. the rna is then re-solubilized in rnase-free water and quantified using a nanodrop. reverse transcription is performed using the retroscript kit, and cDNA is synthesized. the cDNA is then purified and used for qrt-pcr using taqman assay reagents on a step one detection system. the protocol uses β-actin as a housekeeping gene for normalization and embryonic mouse brain as a calibrator. gene expression levels are calculated using the delta delta ct (2^-ΔΔct) method, with results expressed as arbitrary units (au). the relative fold changes compared to wild-type (wt) are indicated for each treatment group. the protocol is open access and distributed under the creative commons attribution license. the authors are michela deleidi, bianca marchetti, federico bertoli, and carmela giachino from various institutions in germany and italy. the protocol was created on march 31, 2023, and is currently in a working status. the protocol is funded by the ASAP (aligning science across parkinson's) grant. the materials include qiazol lysis reagent, rneasy lipid tissue mini kit, nanodrop, retroscript kit, taqman universal pcr master mix, step one detection system, and other reagents. the protocol is intended for use in research on neurodegenerative diseases, particularly those involving mitochondria and inflammation.