RNA isolation from Peyer’s patch lymphocytes and mononuclear phagocytes to determine gene expression profiles using NanoString technology
2Division of Molecular Genetics Wadsworth Center, New York State Department of Health, Albany NY, USA
3Biochemistry and Immunology Core, Wadsworth Center, New York State Department of Health, Albany NY, USA
4Department of Biomedical Sciences, University at Albany, School of Public Health Albany, Albany NY, USA
5Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
- RNase Zap Decontamination Solution (Thermo Fisher Scientific, cat. # AM 9780)
- 70% Ethanol (Pharmco, cat. # 04343-19)
- β-glucan particles (GPs) 
- β-glucan particles with residual mannan content (GMPs) 
- β-Mercaptoethanol (Sigma, cat. # M6250)
- 0.4% Trypan Blue (Amresco, cat. # K940)
- RNeasy Mini Kit (Qiagen, cat. # 74106)
- DNase RNase-Free Set (1500 Kunitz units) (Qiagen, cat. # 79254)
- SUPERase·In RNase Inhibitor (20 U/µl) (Thermo Fisher Scientific, cat. # AM 2694)
- Agilent RNA 600 Pico Kit (Agilent Technologies, cat. # 5067-1513)
- Agilent RNA 600 Nano Kit (Agilent Technologies, cat. # 5067-1511)
- nCounter Low RNA input Amplification kit (Nanostring, cat. # LOW-RNA-48)
- Target enrichment Primer pool (NanoString, cat. # PP-MIP1-12)
- nCounter® PanCancer Immune Profiling Kit (NanoString, cat. # GXA-PATH1-12)
- Rat anti-mouse CD45R/B220 PerCp Clone RA3-6B2 (BD Pharmigen, cat. # 553093)
- Rat anti-mouse CD3 FITC-Clone 17A2 (BD Pharmigen, cat. # 555274)
- Rat anti-mouse Fc-Block CD16/CD32-Clone 2.4G2 (BD Pharmigen, cat. # 553142)
- Armenian Hamster anti-mouse CD11c-Clone N418 (eBioscience, cat. # 17-0114-82)
- Luna Universal qPCR Master Mix (New England Biolabs, cat. # M3003X)
- ProtoScript II First Strand cDNA Synthesis Kit (New England BioLabs, cat. # E6560S)
- DNA Oglios (IDT, # See Fig. S1 for sequences)
- Gold Taq Flexi DNA polymerase (Promega, cat. # M8295)
- Deoxynucleotide (dNTP) Solution Set (New England BioLabs, cat. # N0446S)
- 100 bp DNA Ladder (Thermo Fisher, cat. # 10488058)
- Kleen Guard Gloves (Kimberly Clark, cat. # 57370)
- Straight Surgical Scissor (FST, cat. # 14060-09)
- Curved Surgical Scissor (FST, cat. # 14061-09)
- 70 µm nylon cell strainer (CELLTREAT, cat. # 229483)
- 12 × 75 mm Polystyrene Tubes with Cell Strainer Cap (BD, cat. # 352235)
- Cell Strainer Pestle (CELLTREAT, cat. # 229480)
- Countess Cell Counter (Thermo Fisher Scientific, cat. # C10281)
- Countess Cell Counting Chamber Slides (Thermo Fisher Scientific, cat. # C10312)
- Squared Petri Dishes (Fisher Scientific, cat. # FB0875711A)
- 22 G × 1.5 in blunt-end feeding needle (Popper Scientific, New Hyde Park, NY)
- Fetal Bovine Serum (Thermo Fisher, cat. # A3160401)
- Spin-X Centrifuge Tube 0.22 µm Filter (Costar, cat. # 8161)
- 2% E-gel (Thermo Fisher Scientific, cat. # G501802)
- iBase (Thermo Fisher Scientific, cat. # G6400)
- Freeze Zone 4.5 Benchtop Lyophilizer (Labconco, cat. #7750020)
- 96 Well Thermal iCycler (Biorad, cat. # 170-8740)
- Chip Priming Station (Agilent, cat. # 5065-4401)
- Vortex Mixer (IKA, cat. # 0025001607)
- 16 pin bayonet electrode cartridges (Agilent, cat. #5065-4413)
- PCR Machine DNA Engine Dyad (MJ Research, cat. # PTC-220)
- 2100 Expert Software (Agilent, Version B.02.08.SI648 (SR2))
- nCounter Prep Station Version 18.104.22.168
- nCounter digital analyzer Version 22.214.171.124
- nSolver analysis software 3.0
- FACSAria IIu Flow Cytometry Sorter (BD, cat. # 643895)
- BD FACSDiva Flow Cytometry Sort/Analysis Softer Ware Version 6.1.3 (BD, cat. # 643629)
- BD Falcon 5 ml Polystryrene Round-Bottom Tube with Cell-Strainer Cap (BD, cat. # 32235)
1.Gavage mice with phosphate buffered saline (PBS) or with antigen of interest. In these experiments, we used 1 × 108 highly purified, Saccharomyces cerevisiae derived β-glucan particles (GPs) as a positive control. As a second positive control, 1 × 108 purified β-glucan particles with exposed mannans (GMPs) were used in 200 µl of 1× PBS. Delivery of particles was achieved using a 22 G × 1.5 in blunt-end feeding needle. Both GPs and GMPs are known to stimulate the immune response [19,21].
Isolation of total Peyer’s patch cells from mice
2.Before euthanizing mice, prepare one tube containing 2 ml of Hanks’ balanced salt solution (HBSS) with 50 µl of SUPERase·In RNase inhibitor (20 U/µl), label as tube number 1, and place it on ice. Label one additional tube as number 2, add 2 ml of HBSS with 30 µl of SUPERase·In RNase Inhibitor (20 U/µl) and place on ice.
2.1.Euthanize mice by CO2 asphyxiation, according to institutional guidelines.
2.2.Cleanse the mouse abdomen with 70% ethanol prior to necropsy. Perform a standard necropsy that involves a 1 cm incision using straight surgical grade scissors along the midline beginning about 1.5 cm from the base of the rib cage. Expose the peritoneal cavity and identify the cecum. Snip the terminal small intestine at the ileal-cecal junction, gently remove the small intestine in its entirety, and snip the stomach-ileal junction to release intact intestine.
2.3.Identify individual PPs located on the anti-mesenteric side of the intestine. Mice typically contain 5 to 10 PPs spaced more or less evenly from the duodenum (proximal) to ileum (distal).
2.4.Using curved surgical scissors, gently excise individual PPs and place them immediately in cold HBSS that contains 50 µl of SUPERase·In RNase inhibitor (tube 1). The scissors should be placed curve-side up just above the PP and then gently applied to the tissue. Add RNAase Zap to surgical instruments and then rinse with ethanol between every mouse.
2.5.Decant HBSS in tube 1 and transfer PPs into tube 2 that contains 2 ml of HBSS with 30 µl of SUPERase·In RNase inhibitor.
Staining cells for cell sorting
3.To generate a cell suspension, decant tube 2 into a 70 μm nylon mesh cell strainer that is resting on a petri dish. This setup should be on ice. Grind PPs with pestle or with the back part of a syringe plunger. The 2 ml should yield approximately 1200 µl after grinding the PPs. Transfer the cell suspension from the petri dish into a microfuge tube . If you use RNAlater RNA Stabilizer Reagent instead of SUPERase·In RNase inhibitor, PPs will feel like rubber at this step and will not yield high quality RNA.
3.1.Save 10–15 µl of cells to count them in step 3.5.
3.2.Using a 96-round bottom plate, add 50 µl of cells per well for single color controls. For B-cells, T-cells and DCs that will be sorted, fill ten wells per cell type, with 100 µl of cells per well. Spreading cells into multiple wells prevents cell clumping in subsequent steps.
3.3.Spin the plate for 2 min at 2100 rpm (887× g), to pellet the cells and decant supernatant by inverting the plate.
3.4.Prepare a (1:100) dilution of Rat anti-mouse Fc-block in flow buffer and resuspend cells in 100 µl of this buffer and incubate on ice for 15 min. Place the ice bucket on a rocker during incubation.
3.5.During the incubation step with the Fc block, count viable cells in the suspension by preparing a 1:20 dilution of cells with trypan blue. Add 10 μl of the cell suspension to the countess chamber slides and count cells. 20 PPs should yield approximately 1.5 × 106 cells per ml with a viability range of 90%–98%.
3.6.After the 15 min incubation, spin the plate for 2 min at 2100 rpm (887× g), and decant supernatant by inverting the plate.
3.7.Resuspend the cells in 100 µl of fluorophore-conjugated antibodies added directly to cell suspensions (1:200 for B and T-cells and 1:40 for CD11c+ phagocytes) and incubate on ice for 30 min with continuous rocking.
3.8.Spin the plate for 2 min at 2100 rpm (887× g), and decant supernatant by inverting the plate.
3.9.Wash cells with 200 μl of flow buffer.
3.10.Spin the plate for 2 min at 2100 rpm (887× g), and remove the supernatant by inverting the plate.
3.11.Resuspend the cells from the plate into 3 ml of flow buffer; some of cells will clump. Pass the 3 ml cell suspension through a filtered cap flow tube that contains 1ml of flow buffer resulting in a total of 4 ml of cell suspension for cell sorting.
Cell sorting of B- and T-lymphocytes and CD11c+ phagocytes and RNA isolation
4.Cells were analyzed and sorted on a FACS Aria IIu cell sorter. Prior to the collection of sorted cells, a cell purity test sort should be performed to check that the cell types of interest are free of debris and contamination by other cell types in the sample.
4.1.Sort CD45R/B220+(CD3-/CD11c-) B-cells, CD3+(CD45R/B220-/CD11c-) T-cells and CD11c+(CD45R/B220-/CD3-) CD11c+ phagocytes onto a filtered Spin-X column that is moistened with 100 µl of flow buffer that containing SUPERase·In RNase inhibitor. For PP B and T-cells we sorted 300000 cells. Since CD11c+ phagocytes are the least abundant of the three cell types in PPs, we sorted a maximum of 50000–80000 cells.
4.2.During sorting, centrifuge the Spin-X column every 100000 cells at 2000 rpm for 1 min at room temperature (20°C to 30°C) as this is the maximum volume that the tubes hold during cell sorting. Discard flow buffer through and continue sorting.
4.3.When sorting is finished, centrifuge the Spin-X column at 2000 rpm for 2 min. Place the filter in a new 1.5 ml collection tube. Discard flow buffer through an old collection tube.
4.4.Add 350 µl of Qiagen’s RLT buffer that contains β-Mercaptoethanol (BME) (10 µl of BME in 1 ml of RLT) to the filter, pipette 20–25 times to mix. Place tube on dry ice for 5 min.
4.5.Incubate on rotary spinner for 10 min and vortex at the end of the incubation. The RNA extraction is performed at room temperature (20°C to 30°C).
4.6.Centrifuge the Spin-X column for 2 min at 13400 rpm. Keep flow-through. To obtain a maximum yield of RNA, the flow through is passed a second time unto the filter.
4.7.Transfer the flow-through back onto the filter, and centrifuge for 2 min at 13400 rpm. Keep flow-through.
4.8.Add 100 µl of RNase free H2O to the filter and pipette 20–25 times, and centrifuge for 1 min at 13400 rpm. Keep flow through and discard the filter.
4.9.Add 250 µl 100% ethanol to the tube.
4.10.Transfer the liquid to the RNeasy spin column and pipette 20–25 times, incubate for 5 min on bench top. Centrifuge for 1 min at 13400 rpm. Discard flow-through.
Column DNase digestion
4.11.Add 350 µl buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 13400 rpm to wash the spin column membrane. Discard the flow-through.
4.12.Add 10 µl DNase I stock solution to 70 µl buffer RDD. Vortex to mix and add 80 µl directly to each RNeasy spin column membrane, then incubate on the bench top for 15 min.
4.13.Add 350 µl buffer RW1 directly onto the RNeasy spin column that contains the 80 µl of DNase I and spin for 15 s at ≥ 10000 rpm. Discard the flow-through.
4.14.Add 500 µl buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s 13400 rpm to wash the spin column membrane. Discard the flow-through.
4.15.Add 500 µl buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 13400 rpm to wash the spin column membrane. Discard the flow-through.
4.16.Add another 500 µl buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 13400 rpm to wash the spin column membrane. Discard the flow-through.
4.17.Place the RNeasy spin column in a new 2 ml collection tube and discard the old collection tube containing the flow-through. Spin at 13400 rpm for 1 min to eliminate any possible carryover of buffer RPE.
4.18.Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30 µl of RNase-free water directly to the spin column membrane, incubate for 2 min, then spin at 13400 rpm for 2 min to elute the RNA. Keep flow-through.
4.19.Add 10 µl of RNase-free water directly to the spin column membrane, incubate for 2 min, then spin at 13400 rpm for 2 min to elute the RNA. Keep flow-through. Vortex the 40 µl of collected RNA.
4.20.Sample can be stored short-term at −20°C; for longer storage, store at −80°C.
Lyophilize RNA to concentrate
5.RNA stored at −80°C must be placed on dry ice and the lid of the tube covered with parafilm. Make small holes on the top of the tube using a flame-heated needle and place inside a lyophilization flask. Lyophilize for 3 h. To ensure that the flask remains cold and the RNA remains frozen, place the flask on dry ice while it is attached to the lyophilizer. Resuspend lyophilized RNA in 6 µl of RNase-free water.
Measurement of RNA using bioanalyzer
6.Before use, thaw sample and ladder on ice until fully resuspended. Take 1 µl of RNA sample and ladder and heat-denature at 70°C for 2 min.
6.1.Agilent RNA 6000 Pico kit protocol was followed as per manufacturer’s instructions.
RT-PCR and RT-qPCR assays for detection of gene expression
7.Synthesize the cDNA using ProtoScript II first strand cDNA Synthesis Kit from NEB using 1 ng of RNA for each cell type with random hexamer primers.
7.1.Use gene-specific primers to perform either reverse transcription-PCR (RT-PCR) or quantitative RT-PCR (RT-qPCR) on the cDNA. RT-PCR was performed on cDNA to ensure the amplification efficiency and specificity of the primers. Using both methods, we chose three housekeeping genes from the immune profiling codeset Hprt, Tubb5 and Sf3a3 (Fig. S1).
7.2.RT-PCR conditions: Initial denaturation 94°C for 3 min, 94°C for 20 s, 55°C for 20 s, 72°C for 20 s (30×) and 72°C for 5 min, followed by hold at 4°C. RT-qPCR conditions: Initial denaturation 95°C for 60 s, 95°C for 15 s and 60°C for 30 s (40×), followed by hold at 4°C.
7.3.To estimate the minimum number of cycles needed for cDNA amplification, we used the RT-qPCR assay, using Luna Universal qPCR Master Mix and the primers for the housekeeping genes mentioned in 7.1.
7.4.The estimated number of cycles from the RT-qPCR was used in the nCounter Low RNA Input Amplification assay.
nCounter low RNA input amplification assay
8.Add RNA (2 ng or less) with the provided reagents in the kit; 0.5 µl of 10× RT enzyme mix, 0.5 µl of 10× Primer Mix and bring up to 5 µl with RNase-free water.
8.1.Synthesize cDNA in thermocycler using the following conditions: anneal primer at 25°C for 10 min, first strand cDNA synthesis at 42°C for 60 min, enzyme inactivation for 85°C for 5 min and hold at 4°C.
8.2.Add the following to the cDNA: 1.5 µl 5× dT Amp Master Mix, 1 µl gene specific primers (500 nM per primer) corresponding to the codeset for multiplexed target enrichment. Gently, flick to mix and centrifuge.
8.3.Set up the amplification reaction in thermocycler. Initial denaturation 95°C for 10 min, 95°C for 15 s and 60°C for 4 min, followed by hold at 4°C. Number of cycles needs to be estimated on cell type, as stated in the note.
8.4.Proceed with NanoString hybridization or store the dsDNA at −80°C.
Reading RNA counts with NanoString
9.Thaw the dsDNA if frozen or proceed with hybridization.
9.1.Heat denatures the dsDNA at 95°C for 2 min. Immediately cool on ice.
9.2.Pre-heat PCR thermocycler to 65°C with the lid set to 70°C.
9.3.Thaw reporter code set and capture code set on ice. Mix by flicking or inverting tubes.
9.4.Prepare master mix for the 12 samples. The master mix includes 70 µl of sample hybridization buffer to the provided reporter code set.
9.5.Label strip tubes and cut the strip in the middle to yield 6 tubes each. Aliquot 8 µl of master mix into strip tubes.
9.6.Adjust dsDNA input to 5 µl in dH2O.
9.7.Add dsDNA to the 8 µl of master mix into labeled strip tubes.
9.8.Add 2 µl of capture probe set to every tube.
9.9.Close the tubes and immediately transfer to the 65°C thermocycler set up in step 9.2.
9.10.Incubate in 65°C for 12–30 h.
9.11.Warm sealed cartridge from −20°C to room temperature and sealed reagent plates from 4°C to room temperature (20°C–30°C).
9.12.Centrifuge reagent plates 2000 g for 2 min.
9.13.Follow prep station automated instructions until it requests sample loading. Immediately remove sample from 65°C, spin in Picofuge, carefully remove caps, and load in prep station. Immediately initiate protocol by pressing start.
9.14.Remove cartridge and seal with adhesive tape to prevent evaporation. Tape is provided in kit.
9.15.Take cartridge and place in digital analyzer for RNA copy counts.
9.16.Upload the Reporter Library file in the provided flash drive.
9.17.Create a CDF file with sample name and description and upload into the digital analyzer.
9.18.Insert cartridge with the seal (step 9.14) into the digital analyzer.
9.19.Initiate counts by pressing start.
9.20.When the digital analyzer is complete download your data for analysis.
9.21.Follow instructions on nSolver or self-analyze.
|Fold change||Adjusted P||Fold change||Adjusted P|
|2||Rapid RNA degradation||Adequate amounts of SUPERase·In RNase inhibitor were not added||Add suggested amounts of SUPERase·In RNase inhibitor|
|3||Rapid RNA degradation||PPs were not ground on ice||PPs must always be dissociated on ice|
|3.11||Cells clump before sorting||Small volumes of flow buffer||Start with a minimum of 1–2 ml of flow buffer as cells are filtered through|
|4||Rapid RNA degradation||Harsh cell lysis||Use a Spin-X column not a syringe|
|7.1||NanoString low input kit does not seem to be efficient||Very low levels of RNA||Estimate the minimum number of cycles needed for cDNA amplification, we used the RT-qPCR assay|
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