Jul 07, 2023

RNA isolation and qRT-PCR

DOI
https://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
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DOI: https://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 24, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 82430
Keywords: Primers, SYBR, PCR, ASAPCRN, rna isolation from cultured cell, rna isolation, quantitative rt pcr, pcr this protocol, pcr, cultured cell, qrt, isolation
Funders Acknowledgements:
ASAP
Grant ID: 000580
Abstract
This protocol describes the RNA isolation from cultured cells and quantitative RT PCR.
Attachments
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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
Aspirate media from cells and rinse cells with PBS On ice .

Isolate RNA using RNeasy Micro Plus kit (Qiagen) according to manufacturer’s protocol.
Generate cDNA from 1 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 10 µL SYBR Green Master Mix (BioRad) with 6.78 µL Sterile Water per sample.

Combine 16.78 µL diluted SYBR Green Master Mix with 0.61 µL each of 10 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 2 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.


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.