Upon receiving the PureLink RNA isolation kit, label the box and all of the buffers with the date received. Once all of the spin cartridges in a kit have been used, properly dispose of any leftover buffers and open a new kit. Saving and using old buffers can result in low yields and impurities in the isolated RNA. It is important to label the date that all buffers were opened/prepared and to prepare fresh lysis buffer (addition of 1% 2-mercaptoethanol) immediately before use. Additionally, it is critical to work in a clean space and use filter tips to prevent RNase contamination of reagents and samples. Gloves and safety glasses should be worn at all times during the completion of this protocol. The isolated RNA samples will contain varying amounts of carryover genomic DNA, which can be removed through the inclusion of the DNase treatment steps described below; however, DNase treatment is optional and may be omitted if the downstream detection methods being used do not detect genomic DNA (e.g. qPCR primer/probe sets used in detection are exon-spanning).
Buffer Preparation
1. When using a new kit, add 300 mL of 100% ethanol to Wash Buffer II. Initial and date the label and lid to indicate that ethanol is already added.
2. Prepare fresh Lysis Buffer by aliquoting the amount of Lysis Buffer required for the procedure and adding 2-mercaptoethanol to a final concentration of 1%.
3. Prepare a fresh aliquot of 70% ethanol in RNase-free water on the day of extraction.
4. (OPTIONAL-DNase) Prepare PureLink DNase mixture in a new, clean Eppendorf tube according to the recipe below for the number of samples plus one additional volume:
10X DNase I Reaction Buffer 8 μL
Resuspended DNase (~3U/μL) 10 μL
RNase-Free Water 62 μL
Final Volume 80 μL
Cell Wash and Harvest
pHBEC (Primary) Cells
1. Blot basal surfaces with Kimwipes and transfer inserts into new, clean wells.
2. Carefully aspirate the medium from the apical compartment.
3. Rinse the apical surface with 400 µL (for 24 mm inserts) or 200 µL (for 12 mm and 6.5mm inserts) of PBS. Aspirate the PBS wash.
4. Add 300 µL (for 24 mm inserts), 250 µL (for 12 mm inserts), or 200 µL (for 6.5 mm inserts) of Lysis Buffer (with 1% 2-mercaptoethanol) to the apical surface.
5. Using a cell scraper or wide-bore pipette tip, dislodge cells from the membrane and collect in new, clean Eppendorf tube.
a. NOTE: If samples are particularly viscous, shear cells 3-5 times with either a pipette tip or 1 mL syringe.
6. Proceed to RNA Purification or store samples at -80 oC.
16HBE Cells
1. Blot basal surfaces with Kimwipes and transfer inserts into new, clean wells.
2. Carefully aspirate the medium from the apical compartment.
3. Rinse the apical surface with 1 mL (for 24 mm inserts) or 500 µL (for 12 mm inserts) of PBS. Aspirate the PBS wash.
4. Add 300 µL of Lysis Buffer (with 1% 2-mercaptoethanol), pipette up and down while distributing the Lysis Buffer across the membrane surface, and transfer the lysate from each sample to individual Eppendorf tubes.
a. NOTE: Use cell scrapers to dislodge cells for 24 mm inserts.
5. Proceed to RNA Purification or store samples at -80 °C.
IMR90 Cells/Primary Fibroblasts
1. Carefully aspirate the medium from the basolateral compartment.
2. Add 300 µL (for 6-well) or 200 µL (for 12-well) of Lysis Buffer (with 1% 2-mercaptoethanol), pipette up and down while distributing the lysis buffer across the well surface, and transfer the lysate from each sample to individual Eppendorf tubes.
a. NOTE: Use cell scrapers to dislodge cells for 6 well plates.
3. Proceed to RNA Purification or store samples at -80oC.
RNA Purification
1. Remove samples from -80˚C and thaw on ice. While samples are thawing, begin to sort/label tubes in preparation for RNA isolation protocol.
a. NOTE: thawing samples at room temperature will often result in low A260/A230 ratios in the purified samples, which can negatively impact downstream applications.
2. Add one volume 70% ethanol to each volume of cell homogenate.
3. Vortex samples thoroughly until precipitate is in solution.
4. Transfer up to 700 µL of the sample to spin cartridge.
5. Centrifuge at 12,000 x g for 30 seconds at room temperature. Aspirate the flow-through, and reinsert the spin cartridge into the same collection tube.
6. Repeat steps 3-4 until the entire sample has been processed.
7. Add 350 µL Wash Buffer I to the spin cartridge.
8. Centrifuge at 12,000 x g for 30 seconds at room temperature. Aspirate the flow-through.
a. (OPTIONAL-DNase) Add 80 μL PureLink DNase mix directly onto the surface of the spin cartridge membrane.
b. (OPTIONAL-DNase) Incubate at room temperature for 15 minutes.
c. (OPTIONAL-DNase) Add 350 μL Wash Buffer I to the spin cartridge.
d. (OPTIONAL-DNase) Centrifuge at 12,000 x g for 30 seconds at room temperature. Aspirate the flow-through
9. Place the column into a new collection tube.
10. Add 500 µL of Wash Buffer II (with ethanol added) to the spin cartridge. Invert once to wash any potential residue from spin cartridge lid and let sit for 1 minute.
11. Centrifuge at 12,000 x g for 30 seconds at room temperature. Aspirate the flow-through.
12. Add 500 µL of Wash Buffer II (with ethanol added) to the spin cartridge, close cap and invert tube, and incubate at room temperature for 2 minutes.
a. NOTE: The inversion and incubation step facilitates better A260/A230 ratios.
13. Centrifuge at 12,000 x g for 30 seconds at room temperature. Aspirate the flow-through.
14. Centrifuge the spin cartridge for 4 minutes at 12,000 x g at room temperature to dry the membrane with bound RNA.
15. Discard the collection tube and insert the spin cartridge into a labeled recovery tube.
16. Add RNase-free water to the center of the spin cartridge in the appropriate volume:
a. NHBE and 16HBE cells: 150 µL for 24 mm, 100 µL for 12 mm, 50 µL for 6.5 mm
b. IMR90 and primary fibroblasts: 50 µL for 6-well and 40-50 µL for 12-well
17. Incubate at room temperature for 2 minutes.
18. Centrifuge the spin cartridge for 2 minutes at 12,000 x g at room temperature to elute the RNA from the membrane into the recovery tube.
a. NOTE: If low yield is expected, elute multiple times through the spin cartridge.
19. Discard columns, vortex samples, and short spin for 10 seconds to collect sample in the bottom of the tube.
20. Store purified RNA on ice, vortex, and use 2 µL to quantify and check sample purity using a Nanodrop Spectrophotometer.
21. Proceed with cDNA synthesis (refer to SOP-NHEERL/EPHD/CRB/SDM/2016-008-000) or store samples at -80oC.