Jul 07, 2023

Public workspaceRNA isolation and qRT-PCR

DOI
dx.doi.org/10.17504/protocols.io.261ge3qojl47/v1
  • Narayana Yadavalli1,2,3,4,5,
  • Shawn M. Ferguson1,2,3,4,6,5
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Berrak Ugur
Open access
DOI: dx.doi.org/10.17504/protocols.io.261ge3qojl47/v1
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. RNA isolation and qRT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3qojl47/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: May 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82430
Keywords: Primers, SYBR, PCR, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes the RNA isolation from cultured cells and quantitative RT PCR.
Attachments
724-1818.docx
724-1818.docx
136KB
Materials
Reagents require:

  1. RNeasy Micro Plus kit (Qiagen)
  2. iScript cDNA synthesis Kit (Bio-Rad)
  3. Primers
  4. SYBR Green Master Mix (BioRad)
  5. 96 well plates (BioRad)
  6. Optical Adhesive Covers
RNA isolation and qRT-PCR
RNA isolation and qRT-PCR
Aspirate media from cells and rinse cells with PBS TemperatureOn ice .

Wash
Isolate RNA using RNeasy Micro Plus kit (Qiagen) according to manufacturer’s protocol.
Generate cDNA from Amount1 µg purified RNA using iScript cDNA synthesis Kit (Bio-Rad) according to manufacturer’s protocol.

The iScript cDNA was diluted 1:10 by using sterile water.
Combine Amount10 µL SYBR Green Master Mix (BioRad) with Amount6.78 µL Sterile Water per sample.

Combine Amount16.78 µL diluted SYBR Green Master Mix with Amount0.61 µL each of Concentration10 micromolar (µM) forward and reverse primers per sample. Pipette this mixture into wells of 96-well qPCR plate 2 technical replicates were ran for each sample.

Pipette Amount2 µL of diluted RNA from step 4 in well with SYBR Green Master Mix.

Cover plate with Optical Adhesive Covers (Applied Biosystems).
Spin down plate in tabletop centrifuge.
Run qPCR in CFX96 Real-Time System (BioRad) using the following PCR steps.
PCR cycle.


PCR
Analysis of qRT PCR data
Analysis of qRT PCR data
Calculate the ΔCT values by subtracting respective gene CT value from housekeeping gene GAPDH value.
Followed by calculating 2ΔCT values, normalize the mRNA expression levels for genes of interest to GAPDH and represented relative to control.