qPCR and RT-PCR
Ayse Ulusoy, Rita Pinto-Costa, Angela Rollar, Donato Di Monte, Michael Klinkenberg, Michael Helwig, Shirley Lee
Abstract
The protocol describes the methodology and materials we use in the Di Monte lab for mRNA extraction and q-PCR/or RT-PCR analyses from fixed tissue samples.
Steps
Dissect the dorsal medulla oblongata from paraformaldehyde-fixed coronal sections
of the medulla oblongata.
Extract total RNA using Nucleic Acid Isolation Kit (Ambion), and measure yield using a nanodrop.
Conventional reverse trancription polymerase chain reaction (RT-PCR)
Synthesize cDNA from 100 ng template RNA using SuperScriptVILO Master Mix (Life Technologies) and the suitable primers (20µLfinal volume).
Run a 30-cycle PCR reaction.
Mix the PCR products with 6x sample buffer (New England
Biolabs) and 5% DMSO and load on 2.0% SeaKem agarose gel
(Lonza Bioscience) pre-stained with RedSafe dye (1:20,000, Intron
Biotechnology)
real-time PCR
If necessary, extract RNA from human brain (Agilent Technologies) as a
reference sample.
Prepare a 20µLl reaction mix containing 1 m l
cDNA, Power SYBRGreen (Applied Biosystems) and
primers.
Run the PCR with a real-time PCR machine (e.g., StepOnePlusTM real-time PCR system, Applied
Biosystems) in triplicates.
