Jul 09, 2024

Public workspaceRNA collection, cDNA conversion and qPCR (SH-SY5Y cells)

DOI
dx.doi.org/10.17504/protocols.io.14egn3ymzl5d/v1
  • 1Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase, MD 20815, USA
Stephanie Vrijsen
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DOI: dx.doi.org/10.17504/protocols.io.14egn3ymzl5d/v1
Protocol Citation: Stephanie Vrijsen, Peter Vangheluwe 2024. RNA collection, cDNA conversion and qPCR (SH-SY5Y cells). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3ymzl5d/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: December 06, 2023
Last Modified: July 09, 2024
Protocol Integer ID: 91851
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000458
Abstract
This protocol describes the isolation of RNA from SH-SY5Y cells and the subsequent conversion to cDNA for qPCR.
Materials
  • NucleoSpin RNA plus kit: 740984.50, Macherey-Nagel
  • High-Capacity cDNA Revers Transcription Kit: 4368814, Thermo Fisher Scientific
  • SYBR Green master mix: 04707516001, Roche
RNA collection
RNA collection
Cells were seeded in 10 cm dishes and used for collection when reaching 70-80% confluency.
(e.g. 3 million cells for collection after Duration48:00:00 )

2d
Remove medium.
Wash once with PBS (-/-).
Scrape and collect cells in PBS(-/-).
Spin down cells (450xg, Duration00:05:00 ).

5m
Wash with PBS(-/-).
Spin down cells (450xg, Duration00:05:00 ).
5m
Remove supernatant.
Isolate RNA following the instructions of the NucleoSpin RNA Plus kit (740984.50, Macherey-Nagel).
! Use a separate, desinfected area to isolate RNA. Use filter tips and dedicated pipets for RNA work.
Determine the concentration and the purity of the isolated RNA using a Nanodrop spectrometer.
cDNA conversion
cDNA conversion
Convert RNA to cDNA using the High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific).
Prepare 5 µg RNA in 20 µl total volume (dilute with RNase free water).
Prepare a 2x Mastermix:
AB
Volume (µl)Component
2µl RT buffer
0,8µl dNTPs (100 µM)
2µl random primers (10x)
1µl Multiscribe transcriptase
4,2µl AD
Volumes are given for one sample, multiply according to your number of samples.

Add 10 µl of the mastermix to 10 µl of the RNA dilution.
Perform a quick vortex and spin down using a table-top centrifuge.
Start program for RNA to cDNA conversion:
ABCDE
Step 1Step 2Step 3Step 4
Temp (°C)2537854
Time10 min120 min5 sec∞




qPCR
qPCR
Prepare a serial dilution of the sample that you choose as standard (1/5, 1/25, 1/125, 1/625, 1/3125). Include water as a negative control.
Pipet in duplo in a 96-well plate (5 µl per well).
Prepare a master mix containing per sample:
  • 10 µl SYBR Green master mix (Roche)
  • 1 µl of 5 µM forward primer
  • 1 µl of 5 µM reverse primer
  • 3 µl water
Prepare a ten-fold dilution of the cDNA samples in duplicates (5 µl cDNA per well).
Include a negative control where the cDNA is exchanged by an equivalent volume of water.
Add 15 µl of the master mix to each well.
Cover plate with a film and spin samples down.
Start the qPCR reaction:
  • 95 °C for Duration00:10:00
  • 50 cycles at 95 °C for Duration00:00:10
  • 55 °C for Duration00:00:30
  • 95 °C for Duration00:01:00

Determine a melting curve from 55 to 95 °C.
11m 40s