TITLE: RNA extraction and quantitative PCR to assay inflammatory gene expression
AUTHORS: OLIVIA HARDING, Erika L.F. Holzbaur

[DESCRIPTION]
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 to60 °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   dishes have been transfected with relevant constructs for 1080 min- 1440 min and treated with appropriate small molecules or vehicles. 1080 min- 1440 min before collection, transfect 1.5 µg Parkin and 0.2 µg EGFP-NEMO to 70-80% confluent cells on each   dish. 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 300 min.












[GUIDELINES]
When working with RNA, take caution to keep space clean to avoid sample degradation by  RNases. Clear bench space and wipe with RNaseZap. Change gloves often and wear a mask.
Use new, sterile supplies of pipet tips and tubes.
Since RNA is vulnerable to degradation, proceed through the extraction and reverse synthase procedures on the same day to avoid storing RNA samples.
Day 1, extract RNA and produce cDNA for all samples for all biological replicates. Day 2, carry out PCR reactions for all replicates.

[STEPS]
SECTION: Initial RNA extraction
1. 
 Aspirate media from each dish.
 
 

SECTION: Initial RNA extraction
2. Add 300 µL cold TRIzol per million cells directly onto the cells and pipet up and down to homogenize.
SECTION: Initial RNA extraction
3.  Transfer to 1.5 mL tube.
SECTION: Initial RNA extraction
4. Incubate 5 min, Room temperature.

SECTION: Initial RNA extraction
5. Add 200 µL chloroform per mL TRIzol.

SECTION: Initial RNA extraction
6. Mix by inversion until cloudy homogenous solution.

SECTION: Initial RNA extraction
7.  Incubate 2 min- 3 min at Room temperature.

SECTION: Initial RNA extraction
8. Centrifuge 15 min at 12 x g, 4 °C.
 

SECTION: Initial RNA extraction
9. 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.

SECTION: Initial RNA extraction
10. Add 500 µL isopropanol to aqueous phase per 1 mL TRIzol used.

SECTION: Initial RNA extraction
11.  Incubate 10 min, Room temperature.




SECTION: Initial RNA extraction
12. Centrifuge 10 min, 12 x g at 4 °C.
 

SECTION: Initial RNA extraction
13. Discard supernatant.
SECTION: Initial RNA extraction
14.  Resuspend pellet in 1 mL 75% EtOH per 1 mL Trizol used.

SECTION: Initial RNA extraction
15.  Vortex quickly then centrifuge 5 min 7.5 x g at 4 °C.

SECTION: Initial RNA extraction
16. Discard supernatant.
SECTION: Initial RNA extraction
17. Air dry pellet 5 min- 10 min.
 



SECTION: Initial RNA extraction
18.  Resuspend the pellet in 50 µL RNase free water by pipetting up and down.
 

SECTION: Initial RNA extraction
19. Incubate at 60 °C  10 min- 15 min.
 



SECTION: Initial RNA extraction
20. Measure concentration of RNA with NanoDrop or other.
SECTION: Reverse Transcriptase Reaction to generate cDNA
21. Thaw 5X first-strand buffer and  0.1 Molarity (M) DTT at Room temperature  immediately before use. Refreeze immediately after.
SECTION: Reverse Transcriptase Reaction to generate cDNA
22. Calculate the volume of each sample needed for 5 µg.

SECTION: Reverse Transcriptase Reaction to generate cDNA
23. To 5 µg RNA, add 1 µL 10 millimolar (mM) dNTP Mix (equal parts each base), 1 µL of oligo(dT)20 (50 micromolar (µM)); and sterile water to 13 µL.

SECTION: Reverse Transcriptase Reaction to generate cDNA
24. Heat at 65 °C, 5 min.
 



SECTION: Reverse Transcriptase Reaction to generate cDNA
25.  Incubate on ice  1 min.

SECTION: Reverse Transcriptase Reaction to generate cDNA
26. Briefly centrifuge.
SECTION: Reverse Transcriptase Reaction to generate cDNA
27. Add 4 µL First-strand buffer, 1 µL 0.1 Molarity (M) DTT, 1 µL  RNase OUT inhibitor, 1 µL SuperScript III.

SECTION: Reverse Transcriptase Reaction to generate cDNA
28. Gently pipet up and down to mix.
SECTION: Reverse Transcriptase Reaction to generate cDNA
29.  Incubate at 50 °C for 45 min.
 

SECTION: Reverse Transcriptase Reaction to generate cDNA
30.  Inactivate by heating to 70 °C for 15 min.

SECTION: Reverse Transcriptase Reaction to generate cDNA
31. The result is cDNA.
SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
32.  Add 5 µL 0.5 Molarity (M) EDTA and 5 µL 1 Molarity (M) NaOH to each, mix by inversion.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
33. Heat at 65 °C 15 min.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
34. Adjust volumes to 50 µL with water.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
35.  Add 100 µL Oligo Binding Buffer to each 50 µL.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
36. Add 400 µL ethanol and mix briefly by pipetting. Transfer to Zymo-Spin Column in the kit.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
37.  Centrifuge 10 x g, 30 s, Room temperature and discard the flow through.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
38. Add 750 µL DNA Wash Buffer to the column.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
39. Centrifuge 10 x g, 30 s, Room temperature. and discard the flow through.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
40. Centrifuge max speed, 1 min, Room temperature.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
41. Transfer the column to a new clean tube and add 15 µL water to the matrix.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
42. Centrifuge at 10 x g, 30 s,  Room temperature  to elute.

SECTION: Clean cDNA (EDTA/NaOH and Zymo Oligo Clean &amp; Conc. Kit)
43.  Measure 260/280 for final conc. The product can be saved at -20 °C.

SECTION: Set up PCR Reactions
44. 
 

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) * 11 µL = ____264______(V) = volume for each set of cDNA.

 
SECTION: Set up PCR Reactions
45. 
 

Mix these then centrifuge quickly.
SECTION: Set up PCR Reactions
46. Split into __8____(p) tubes > (____3____(n) * 10 µL = ____30_____(Pinitial)) in each tube.

SECTION: Set up PCR Reactions
47. Add 0.33 µL * n = ___1_____ uL each primer (10 micromolar (µM)) respectively to get total ____32_____(~Pfinal uL)/tube.

SECTION: Set up PCR Reactions
48. Mix again, centrifuge, and add 10 µL each reaction to wells.

SECTION: Set up PCR Reactions
49. Seal the plate with an adhesive cover then centrifuge to get rid of air bubbles and ensure components are combined.
SECTION: Set up PCR Reactions
50. Can store this at Room temperature  1440 min.

SECTION: Set up PCR Reactions
51. Run the reaction in the QuantStudio with the following procedure.