Aug 02, 2023

Public workspaceqPCR and RT-PCR

DOI
dx.doi.org/10.17504/protocols.io.j8nlkwbewl5r/v1
  • 1DZNE
Ayse Ulusoy
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkwbewl5r/v1
Protocol Citation: Ayse Ulusoy, Michael Klinkenberg, Michael Helwig, Angela Rollar, Shirley Lee, Rita Pinto-Costa, Donato Di Monte 2023. qPCR and RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwbewl5r/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: August 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69173
Keywords: ASAPCRN
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.
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)
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 (Amount20 µL final 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
real-time PCR
If necessary, extract RNA from human brain (Agilent Technologies) as a
reference sample.
Prepare a Amount20 µL l reaction mix containing 1 ml
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.