RNA collection, cDNA conversion and qPCR (SH-SY5Y cells)
Peter Vangheluwe, Stephanie Vrijsen
Abstract
This protocol describes the isolation of RNA from SH-SY5Y cells and the subsequent conversion to cDNA for qPCR.
Steps
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 48h 0m 0s)
Remove medium.
Wash once with PBS (-/-).
Scrape and collect cells in PBS(-/-).
Spin down cells (450xg, 0h 5m 0s).
Wash with PBS(-/-).
Spin down cells (450xg, 0h 5m 0s).
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
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:
| A | B |
|---|---|
| Volume (µl) | Component |
| 2 | µl RT buffer |
| 0,8 | µl dNTPs (100 µM) |
| 2 | µl random primers (10x) |
| 1 | µl Multiscribe transcriptase |
| 4,2 | µl 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:
| A | B | C | D | E |
|---|---|---|---|---|
| Step 1 | Step 2 | Step 3 | Step 4 | |
| Temp (°C) | 25 | 37 | 85 | 4 |
| Time | 10 min | 120 min | 5 sec | ∞ |
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
0h 10m 0s - 50 cycles at 95 °C for
0h 0m 10s - 55 °C for
0h 0m 30s - 95 °C for
0h 1m 0s
Determine a melting curve from 55 to 95 °C.
