Sep 29, 2025

Public workspaceRNA isolation and qRT-PCR

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l22r9pl1y/v1
  • Khaja Mohieddin Syed1,2,
  • Dirk Hockemeyer1,2
  • 1University of California, Berkeley, Berkeley, CA 94720, USA;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 2081
Oriol Busquets
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l22r9pl1y/v1
Protocol Citation: Khaja Mohieddin Syed, Dirk Hockemeyer 2025. RNA isolation and qRT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22r9pl1y/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: January 15, 2024
Last Modified: September 29, 2025
Protocol Integer ID: 93519
Keywords: ASAPCRN, procedure for rna isolation, rna isolation, pcr primer sequence table, pcr this protocol, cdna synthesis, qrt, pcr
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000486
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-024409
Abstract
This protocol describes the procedure for RNA isolation, cDNA synthesis and qRT-PCR.

Protocol overview
A. RNA isolation and cDNA synthesis
B. qRT-PCR

qRT-PCR primer sequence table
Gene NameForward Primer Sequence (5’-3’)Reverse Primer Sequence (5’-3’)
POU5F1ATTCTCCAGGTTGCCTCTCAGTGGAGGAAGCTGACAACAA
NANOGAGTCCCAAAGGCAAACAACCCACTTCTGCTGGAGGCTGAGGTATTTCTGTC
SOX2TTCACATGTCCCCGAACTACCAGATCACATGTGTGAGAGGGGCAGTGTGC
FOXA2GGGGTAGTGCATCACCTGTTCCGTTCTCCATCAACAACCT
LMX1AACGTCCGAGAACCATCTTGACCACCACCGTTTGTCTGAGC
NR4A2GCTGGACTCCCCATTGCTTTCGGAGCTGTATTCTCCCGAA
KCNJ6GCTACCGGGTCATCACAGATACTGCATGGGTGGGAAAAGAC
PITX3CCAACCTTAGTCCGTGCCAGTGTGTAGGGCCTAGTCCACC
THCCGAGCTGTGAAGGTGTTTGACGGGCCGGGTCTCTAGAT
EN2AGGAGCTGAGCCTCAACGAGTCCTTGGCTGTGGTGGAGTGGTTG
AADCGAACAGACTTAACGGGAGCCTTTAATGCCCGGTAGTCAGTGATAAGC
SYN1GCAAGGACGGAAGGGATCACATCACCTGAGCCATCTTGTTGACCACGA
GAPDHTCGGAGTCAACGGATTTGGTCCTGGAAGATGGTGATGGGA

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RNA Isolation and qR...
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Troubleshooting
RNA Isolation and cDNA Synthesis
RNA was isolated using the RNeasy Kit (Qiagen, Cat#74014) according to manufacturer instructions.
Total of Amount2 µg RNA was used for cDNA synthesis and prepared using high capacity reverse transcriptase kit (ThermoFisher Scientific Cat#4368814) as per the manufacturer's protocol below.

ComponentVolume per reaction, µL (1X)
100mM dNTPs0.8
RNAse Inhibitor1
Multiscribe Reverse Transcriptase1
10X Random Primer2
10x RT buffer2
Nuclease free water3.2

Amount2 µg of RNA was used in the reaction mix. Nuclease free water was added for a total reaction volume of 20ul. The below program was used for cDNA synthesis on the standard thermocycler.

TemperatureTime (min)
25℃10
37℃120
85℃5
4℃Hold

The cDNA samples were diluted at 1:5 with nuclease free water before using them for qRT-PCR. Amount2 µL of the cDNA sample was used per reaction/well to check the expression of specific genes such as POU5F1, NANOG, TH, and KCNJ6.

qRT-PCR
Real time qRT-PCR was performed using PowerUP SYBR green (ThermoFisher Scientific, Cat#A25741) on a QuantStudio 6 flex PCR machine with 384 well plate format. Sample reactions were prepared as per reaction master mix below.

ComponentsVolume per reaction, µL (1X)
2x SYBR green5
Forward primer (10µM)1
Reverse Primer (10µM)1
cDNA2
Nuclease free water1

Reaction mix was vortexed and briefly spun down before loading Amount10 µL of master mix per well.

Standard 40 cycle program was used on the QuantStudio 6 flex for the thermocycler reaction.
Results (ct values) were normalized to GAPDH.