RNA extraction and quantitative PCR to assay inflammatory gene expression
OLIVIA HARDING, Erika L.F. Holzbaur
RNA extraction
Reverse transcription
cDNA
Polymerase chain reaction (PCR)
Quantitative realtime PCR
Gene expression
ASAPCRN
Abstract
Real-time quantitative PCR (RT-qPCR) is a sensitive assay to determine the production of selected mRNA transcripts in various conditions. We required such an assay to demonstrate the effects of mitochondrial depolarization in the presence of Parkin, since we found that damaged mitochondria recruited the NF-kB effector complex molecules, NEMO and IKKb. We developed this protocol to test levels of NF-kB response genes in a cell model transiently over-expressing Parkin. With this technique we found significant upregulation of key pro-inflammatory genes normalized to a housekeeping gene, Gapdh.
Before start
- Set one heat source to
60°C. - Set one heat source to
50°C. - Prepare 75% ethanol with RNase/DNase free water
- The start point for this protocol is after cells grown on
6 cmdishes have been transfected with relevant constructs for18h 0m 0s-24h 0m 0sand treated with appropriate small molecules or vehicles.18h 0m 0s-24h 0m 0sbefore collection, transfect1.5µgParkin and0.2µgEGFP-NEMO to 70-80% confluent cells on each6 cmdish. These should yield ~1 million cells per dish - For each replicate, one dish was treated with AntA/OligA, one dish was treated with TNFa (positive control), and one dish was treated with vehicle (control) for
5h 0m 0s.
Attachments
Steps
Initial RNA extraction
Aspirate media from each dish.
Add 300µL cold TRIzol per million cells directly onto the cells and pipet up and down to homogenize.
Transfer to 1.5 mL tube.
Incubate 0h 5m 0s, Room temperature.
Add 200µL chloroform per mL TRIzol.
Mix by inversion until cloudy homogenous solution.
Incubate 0h 2m 0s- 0h 3m 0s at Room temperature.
Centrifuge 0h 15m 0s at 12x g,0h 0m 0s, 4°C.
Transfer aqueous phase (top) containing RNA to new tube by angling at 45°C and carefully pipetting out. The other phases can be saved for protein or DNA isolation.
Add 500µL isopropanol to aqueous phase per 1mL TRIzol used.
Incubate 0h 10m 0s, Room temperature.
Centrifuge 0h 10m 0s, 12x g,0h 0m 0s at 4°C.
Discard supernatant.
Resuspend pellet in 1mL 75% EtOH per 1mL Trizol used.
Vortex quickly then centrifuge 0h 5m 0s 7.5x g,0h 0m 0s at 4°C.
Discard supernatant.
Air dry pellet 0h 5m 0s- 0h 10m 0s.
Resuspend the pellet in 50µL RNase free water by pipetting up and down.
Incubate at 60°C 0h 10m 0s- 0h 15m 0s.
Measure concentration of RNA with NanoDrop or other.
Reverse Transcriptase Reaction to generate cDNA
Thaw 5X first-strand buffer and 0.1Molarity (M) DTT at Room temperature immediately before use. Refreeze immediately after.
Calculate the volume of each sample needed for 5µg.
To 5µg RNA, add 1µL 10millimolar (mM) dNTP Mix (equal parts each base), 1µL of oligo(dT)20 (50micromolar (µM)); and sterile water to 13µL.
Heat at 65°C, 0h 5m 0s.
Incubate On ice 0h 1m 0s.
Briefly centrifuge.
Add 4µL First-strand buffer, 1µL 0.1Molarity (M) DTT, 1µL RNase OUT inhibitor, 1µL SuperScript III.
Gently pipet up and down to mix.
Incubate at 50°C for 0h 45m 0s.
Inactivate by heating to 70°C for 0h 15m 0s.
The result is cDNA.
Clean cDNA (EDTA/NaOH and Zymo Oligo Clean & Conc. Kit)
Add 5µL 0.5Molarity (M) EDTA and 5µL 1Molarity (M) NaOH to each, mix by inversion.
Heat at 65°C 0h 15m 0s.
Adjust volumes to 50µL with water.
Add 100µL Oligo Binding Buffer to each 50µL.
Add 400µL ethanol and mix briefly by pipetting. Transfer to Zymo-Spin Column in the kit.
Centrifuge 10x g,0h 0m 0s, 0h 0m 30s, Room temperature and discard the flow through.
Add 750µL DNA Wash Buffer to the column.
Centrifuge 10x g,0h 0m 0s, 0h 0m 30s, Room temperature. and discard the flow through.
Centrifuge max speed, 0h 1m 0s, Room temperature.
Transfer the column to a new clean tube and add 15µL water to the matrix.
Centrifuge at 10x g,0h 0m 0s, 0h 0m 30s, Room temperature to elute.
Measure 260/280 for final conc. The product can be saved at -20°C.
Set up PCR Reactions
| A | B | C | D | E |
|---|---|---|---|---|
| Sample SYBR | SYBR Master Mix | Fwd and Rev Primers (10 uM stock to 300 nM final) | cDNA (1:100 dilutions) | Nuclease free water (to 44 uL) |
| For one reaction (total 11 uL) | 5.5 uL | 0.33 uL | 11 ng (this is themaximum mass) | varying |
We use the following worksheet to plan volumes needed for each reaction.
The following is our example.
Number of different primer sets = _8(p)
Number of replicates per primer set = 3___(n).
8(p) * 3___(n) = 24_(T) = number of reactions per cDNA sample.
24__(T) * 11µL = 264__(V) = volume for each set of cDNA.
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Replicate | Sample | SYBR Master Mix (V / 2) | cDNA (11 * T ug) | Nuclease free water V – (0.33*n) – (V/2) – cDNA volume | Fwd and Rev Primers (10 uM stock to 300 nM final) (0.33 uL * n) add later |
| N1 | No template control | 132 | - | 130 | 1 of each |
| veh | 132 | 5.2 | 124.8 | 1 of each | |
| TNF | 132 | 3.5 | 126.5 | 1 of each | |
| AO | 132 | 4.5 | 125.5 | 1 of each | |
| N2 | No template control | 132 | - | 130 | 1 of each |
| veh | 132 | 4.08 | 125.9 | 1 of each | |
| TNF | 132 | 2.1 | 127.9 | 1 of each | |
| AO | 132 | 2.07 | 127.9 | 1 of each | |
| N3 | No template control | 132 | - | 130 | 1 of each |
| veh | 132 | 3.22 | 126.7 | 1 of each | |
| TNF | 132 | 4.88 | 125.1 | 1 of each | |
| AO | 132 | 2.18 | 127.8 | 1 of each |
Mix these then centrifuge quickly.
Split into 8__(p) tubes > (3(n) * 10µL = 30_(Pinitial)) in each tube.
Add 0.33µL * n = 1__ uL each primer (10micromolar (µM)) respectively to get total 32_(~Pfinal uL)/tube.
Mix again, centrifuge, and add 10µL each reaction to wells.
Seal the plate with an adhesive cover then centrifuge to get rid of air bubbles and ensure components are combined.
Can store this at Room temperature 24h 0m 0s.
Run the reaction in the QuantStudio with the following procedure.
| A | B | C | D |
|---|---|---|---|
| Step | Temp (C) | Duration | Cycles |
| Cycling Mode | |||
| UDG activation | 50 | 2 min | - |
| Dual Lock DNA polymerase | 95 | 2 min | - |
| Denature | 95 | 15 sec | 40 |
| Anneal | 56* | 15 sec | |
| Extend | 72 | 1 min | |
| Dissociation curve | |||
| 1 | 1.6C/sec to 95 | 15 sec | - |
| 2 | 1.6C/sec to 60 | 1 min | - |
| 3 | 0.15C/sec to 95 | 15 sec | - |
