TITLE: RNA Extraction and RT-qPCR of Human Lung Organoids 
AUTHORS: Morris Baumgardt, Maren Hülsemann, Maren Mieth, Doris Frey, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke

[DESCRIPTION]
This protocol describes the RNA extraction from human alveolar-like organoids followed by the perfomance of a RT-qPCR to quantify the amount of hostfactors (ACE2, TMPRSS2, and FURIN) of SARS-CoV-2. The protocol contains detailed steps for the lysis of human alveolar-like organoids, as wells as the RNA extraction followed by the reverse transcription into cDNA. In the next section the protocol describes in detail how to quantify the gene expression with the TaqMan gene expression kit to obtain donor dependent Ct values. 

[BEFORE_START]
To avoid contaminations RNAase/DNAase free reagents, consumables and filter tips must be used at all steps.
Please use RNA low bind tubes.

[GUIDELINES]
This protocol describes the processing of human alveolar-like organoids which have been grown according to Youk et al., 2020. https://doi.org/10.1016/j.stem.2020.10.004.

[STEPS]
SECTION: Organoid lysis for RNA isolation
1. Grow your 3D model as described.
 

SECTION: Organoid lysis for RNA isolation
2. Remove the entire organoid medium from the wells.
SECTION: Organoid lysis for RNA isolation
3. Add 1 mL ice-cold base medium and collect Cultrex with organoids in a 2 mL tube and flush well with additional  1 mL base medium and collect also.

SECTION: Organoid lysis for RNA isolation
4. To disolve the Cultrex place the tube at 4 °C for 5 min.

SECTION: Organoid lysis for RNA isolation
5. Centrifuge at 900 x g, 5 min, 4 °C.

SECTION: Organoid lysis for RNA isolation
6. Carefully remove supernatant and add 350 µL RLT Puffer + β-Mercaptoethanol to the organoids (pellet).
SECTION: Organoid lysis for RNA isolation
7. Place supernatant in a 1.5 mL safelock tube.

 

Isolate RNA using „RNeasy Mini Kit” from Qiagen (Cat No./ID 74104).
 

SECTION: Organoid lysis for RNA isolation
8. After isolation, measure RNA quality via RIN (RNA Integrity Number) using 2100 Bioanalyzer instrument (manufacturer's instructions, no changes have been made to the protocol included in the kit "Agilent RNA 6000 Nano Kit"). High-quality RNA will contain an RIN of at least 8. All samples used in this protocol had a RIN of 10.

  

SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
9. All steps on ice.

SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
10. To quantify the amount of RNA before reverse transcription use 1.5 µL of your RNA solution using the NanoDrop.

SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
11. Allow the kit components to thaw on ice (high capacity cDNA reverse transcription kit, Applied biosciences).
SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
12. Calculate the volume of components needed to prepare the required number of reactions.
SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
13. Prepare the RT master mix on ice.
SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
14. One reaction needs:

 





SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
15. According to NanoDrop-measurement prepare an RNA/water solution containing 1 µg RNA/10 µL water in a thin wall PCR tube. If RNA concentration is low, 500 ng RNA/10 µL water is also sufficient.

SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
16. Include RT control: use water instead of RNA, run one control with each primer.
SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
17. Add 10 µL of 2x reverse transcription master mix to each tube.

SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
18. Use the following program for reverse transcription:


 

SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
19. Add 80 µL H2O to each tube (cDNA).

SECTION: Preparation of 2x RT master mix and cDNA reverse transcription
20. Use the cDNA directly for qPCR or store at -20 °C.

SECTION: TaqMan - quantitative PCR
21. Prepare the qPCR master mix. Calculate the volume of components needed to prepare the required number of reactions for each Gene expression assay.

 

 
SECTION: TaqMan - quantitative PCR
22. Perform qPCR, pipetting 15 µL of respective TaqMan gene expression assay master mix to the bottom of the well, then pipet 5 µL of cDNA on the upper wall of the well.

SECTION: TaqMan - quantitative PCR
23. Seal the plate with a Clear Adhesive Film.
SECTION: TaqMan - quantitative PCR
24. Centrifuge the plate for 1200 x g, 30 s.

SECTION: TaqMan - quantitative PCR
25. Important quality controls:
Run each sample in duplicates.
Always run house keeping genes (e.g., GAPDH and ß-ACTIN) and gene of interest on the same plate.
Include water control for each primer.
Include RT control from previous step.
SECTION: TaqMan - quantitative PCR
26. Run the qPCR reaction in a Thermal Cycler: 

 

SECTION: Analysis
27. Exemplary result: