CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS
1. Upon receiving the ZYMO Quick-RNA miniprep kit, label the box and all of the buffers with the date received.
2. Saving and using old buffers can result in low yields and impurities in the isolated RNA. Once all of the spin cartridges in a kit have been used, properly dispose of any leftover buffers and open a new kit. The use of older wash buffer results in lower RNA yields.
3. It is critical to work in a clean space and use filter tips to prevent RNase contamination of reagents and samples.
4. Gloves and safety glasses should be worn at all times during the completion of this protocol.
5. The isolated RNA samples will contain varying amounts of carryover genomic DNA (gDNA), which can be removed through the inclusion of the DNase treatment steps described in the referenced manual; however, DNase treatment is optional and may be omitted unless downstream uses of the isolated RNA are sensitive to the presence of gDNA (e.g., qPCR with primer/probe sets that are not exon-spanning).
6. Carryover and contamination of purified samples with guanidine from the lysis and RNA prep buffers can occur easily and will result in low A260/A230 ratios and impact downstream uses of the purified RNA. There are notes, as appropriate, to reduce guanidine contamination in the protocol below.
Quality Control:
Purified RNA samples will be analyzed by spectrophotometry for absorbance at wavelengths of 260 nm (A260), 280 nm (A280), and 230 nm (A230) as indicators of nucleic acid, protein (via aromatic amino acids), and organic compound/chaotropic salts, respectively. Ideally, samples will have an A260/A280 ratio of approximately 2.0, which represents a lack of both RNA degradation and excessive protein carryover from the purification. Further, samples will ideally have an A260/A230 ratio of greater than 1.8. Samples with A260/A230 ratios below 1.8 may limit RNA sample performance in downstream analyses. The A260/A280 and A260/A230 ratios for each sample should be recorded. For high-cost and/or high-sensitivity downstream applications, RNA samples should be evaluated by an Agilent 2100 bioanalyzer.
Reagent Preparation
1. When opening a new kit, add 96 mL of 100% ethanol to RNA Wash Buffer using a 50 mL pipet. Initial and date the label and lid to indicate that ethanol is already added.
Sample Preparation
1. Aspirate medium from adherent cell monolayer.
2. Add RNA lysis buffer directly to the cell monolayer.
· NOTE: Add 300 µL of RNA lysis buffer using a P1000 pipette to cell monolayers of ≧ 5 x 106 cells.
i. NOTE: This volume of RNA lysis buffer is sufficient for the 12 mm Transwell® inserts and 12 well plates.
· NOTE: Add 600 µL of RNA lysis buffer using a P1000 pipette to cell monolayers of 5 x 106 – 107 cells.
i. NOTE: This volume of RNA lysis buffer is sufficient for the 24 mm Transwell® inserts and 6 well plates.
3. Briefly tilt the plate back and forth to facilitate lysis buffer interaction with the entire cell monolayer.
4. Transfer lysed cells with a P1000 pipette to an RNase-free Eppendorf tube.
5. Freeze samples in a -80 ˚C until ready to purify.
Sample Thawing and Preparation for Isolation
1. Remove samples from -80 ˚C and thaw at room temperature for 10-20 minutes or until samples have thawed entirely and appear clear. While samples are thawing, begin to sort/label tubes into conical tubes in preparation for RNA isolation protocol.
a. NOTE: Appropriate amounts, depending on the sample number, of the RNA lysis reagents, 100% Ethanol, RNA Prep Buffer, and RNA Wash Buffer, can be aliquoted into conical tubes while samples thaw to facilitate the ease of downstream steps and minimize the possibility of contamination of the RNA lysis reagent stocks.
b. NOTE: While thawing samples at room temperature will often result in low A260/A230 ratios in the purified samples when using other RNA isolation kits; however, thawing at room temperature is associated with higher ratios with this kit.
c. NOTE: When proceeding through RNA purification, open tubes with two gloved hands. Opening the cap with your thumb of the hand that is holding the tube increases the chance of transferring guanidine residue from lysis buffer into the sample.
RNA Purification
NOTE: For Steps 1 – 10 use a P1000 pipette for all volume transfers.
1. Add one volume (equal to the volume of lysis buffer used during sample preparation) of 100% ethanol to each volume of cell homogenate in lysis buffer.
2. Vortex samples thoroughly (approximately 4 seconds) until ethanol is in solution.
3. Transfer the mixture to a Zymo-Spin™ IIICG Column1 (green) in a collection tube.
a. NOTE: Transfer up to 700 µL of the sample to spin cartridge at a time.
4. Centrifuge at 13,000 x g for 30 seconds at room temperature. Aspirate the flow-through and reinsert the spin cartridge into the same collection tube.
a. NOTE: Do not decant the flow through. Doing so generally results in guanidine contamination of the samples.
b. If the sample volume exceeds 700 µL then repeat steps 4-5 until the entire sample has been run through the column.
5. Add 400 µL RNA Prep Buffer to the column. Incubate at room temperature for 1 minute.
6. Centrifuge at 13,000 x g for 30 seconds at room temperature. Aspirate the flow-through.
a. NOTE: Do not decant the flow through. Doing so generally results in guanidine contamination of the samples.
7. Add 700 µL of Wash Buffer (with ethanol added) to the spin cartridge. Incubate at room temperature for 1 minute.
8. Centrifuge at 13,000 x g for 30 seconds at room temperature. Aspirate the flow-through.
9. Add 400 µL of Wash Buffer (with ethanol added) to the spin cartridge. Incubate at room temperature for 1 minute.
10. Centrifuge at 13,000 x g for 2 minutes at room temperature. Aspirate the flow-through.
11. Discard the collection tube and flow through then insert the spin cartridge into a labeled recovery tube.
a. NOTE: Proceed to the elution step immediately after transferring all of the columns in the sample set. Yield will be reduced if the RNA dries on the column before the addition of water in the elution step.
12. Add RNase-free water to the center of the spin cartridge using a P200 pipette:
a. H441 cells:
· 100 µL for 24mm Transwell® insert
· 50 µL for 12mm Transwell® insert
b. HULEC and primary endothelial cells:
· 100 µL for 6 well
· 50 µL for 12 well
c. NOTE: To obtain a more concentrated RNA elution use ≥ 50 μL elution; however, note that this method will generally result in a lower total RNA yield.
13. Incubate at room temperature for 1 minute.
14. Centrifuge the spin cartridge for 30 seconds at 13,000 x g at room temperature to elute the RNA from the membrane into the recovery tube.
15. Discard columns, vortex samples, and short spin for 5 seconds to collect sample in the bottom of the tube.
16. Store purified RNA on ice and use a P2 pipette to quantify 2 µL of sample and to check sample purity using a Nanodrop spectrophotometer.
17. Record RNA concentration, A260/A280 ratio, and A260/A230 ratio.