Jun 13, 2023
Gene Expression
- michela.deleidi1,2,
- Bianca Marchetti3,4,
- Federico Bertoli1,2,
- Carmela Giachino3
- 1Mitochondria and Inflammation in Neurodegenerative Diseases, DZNE, Tübingen-Germany;
- 2Hertie Institute for Clinical Brain Research, University of Tübingen;
- 3Neuropharmacology Laboratory, Oasi Research Institute-IRCCS, Troina, Italy;
- 4Biomedical and Biotechnological Sciences, Pharmacology Section, University of Catania-Italy
Protocol Citation: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino 2023. Gene Expression. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jyr8lo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 31, 2023
Last Modified: June 13, 2023
Protocol Integer ID: 79817
Keywords: RT-PCR, quantify gene expression
Funders Acknowledgements:
ASAP-Aligning Science Across Parkinson’s
Grant ID: ASAP-000420
Abstract
RT-PCR is a technique to quantify gene expression in samples.
Attachments
k4drb48if.docx
14KB
Materials
Materials
QIAzol Lysis Reagent (200ml)QiagenCatalog #79306
RNeasy Lipid Tissue Mini Kit (50)QiagenCatalog #74804
- Nanodrop-ND 1000
- Retroscript Kit (Ambion #1710, Austin, Texas)
- 2X TaqMan Universal PCR Mastrer Mix (Applied Biosystems)
- Step One Detection System (Applied Biosystems)
- lysis buffer (Qiagen)
- DNase I, RNase-free (Qiagen)
- NanoDrop ND-100 (Nano Drop Technologies)
- the Retroscript Kit (Ambion)
- QIAquick PCR Purification kit (Qiagen)
Gene Expression
Gene Expression
Perform gene expression on tissue samples of the ventral midbrain (containing the substantia nigra pars compacta, SNpc).
RNA Extraction
RNA Extraction
At due time points, homogenize tissue samples in 1 mL 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 30 µL of RNase-free water and determine their concentrations spectrophotometrically by A260 (Nanodrop-ND 1000).
Reverse Transcription
Reverse Transcription
Perform cDNA synthesis using Retroscript Kit (Ambion #1710, Austin, Texas) according the manufacturer’s instructions.
Add 50 µL of water solution containing 0.5 µg of each pool to an equal volume of 2X TaqMan Universal PCR Mastrer Mix (Applied Biosystems).
Real Time PCR
Real Time PCR
Perform real-time quantitative PCR using TaqmanTM Assay 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 2 µg of total RNA using the Retroscript Kit (Ambion).
After purification using QIAquick PCR Purification kit (Qiagen), use 250 ng 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.
Note
We used the housekeeping gene, β-actin, as normalizer and embryonic mouse brain as calibrator.
Using the delta delta Ct (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.