Welcome to the Integrated Department of Immunology at the University of Colorado - School of Medicine and National Jewish Health.

• Sort cells by flow cytometry, pellet at 350 xg for 20 mins, aspirate off supernatant, and resuspend in freshly mixed 1mL RLT plus (Qiagen #1053393) + 10uL BME
• Mix well by vortexing at maximum speed for 0.5 – 1 min
• Quick spin to get sample back to bottom of the tube
• If necessary, can store here at -80˚C. However, it is optimal to isolate RNA fresh. If freeze-down, do quick thaw at 37˚C and mix well again by vortexing at maximum speed for 0.5 – 1 min
• If continuing forward:
• RNase-zap work area (Argos #D6002), reagents, and gloves
• Bring the RNeasy miniElute column (Qiagen #74204) to room temperature
•  Add to QIA shredder (Qiagen #79654) to homogenize and spin at maximum speed 2.5 mins at room temperature.
• If centrifuge has a “soft” mode, turn OFF as many of the subsequent spins are very short and this helps get rid of contaminants
• Transfer to new tube. Avoid cellular debris pelleted during spin above.
• Use low-binding tubes (Eppendorf #022431021) and tips to minimize RNA loss during isolation
•  Add 70% EtOH 1:1 (Fisher 200 proof molecular biology grade #BP2818-500) and shake vigorously ~15 seconds to mix
• Make 70% EtOH fresh
• Minimize vortex steps to avoid shearing DNA (and long non-coding RNA if interested) as this makes contaminating DNA harder to detect
• Quick spin to get sample back to bottom of the tube
• Transfer samples to Qiagen RNeasy MiniElute column
• Preferred because of ability to DNase treat on the column, small elution volume, and subsequent higher RNA concentration
• Spin at 10,000 xg 1 min at room temperature
• Discard flow through and repeat two steps above for remainder of samples to run through the same column
• Aspirate flow through (vs. decant) to reduce salt contamination on outside of RNeasy column that can contaminate sample later upon elution
• DNase treat on column (Qiagen RNase free DNase set recommended by RNeasy column packet #79254):
• Wash with 350 uL RWI from the Qiagen RNeasy MiniElute kit at 10,000 xg for 15 seconds at room temperature
• Add 10 uL DNase + 70 uL RDD mixed by gentle inversion if any. DNase very sensitive to physical denaturation –do not vortex or pipet up and down to mix
• Incubate at room temperature 15 minutes
• Wash by adding 350 uL RWI (Qiagen #1014567) at 10,000 xg for 15 seconds
• If necessary, repeat DNase treatment or increase incubation time to 20 mins
• Put column into new wash tube to prevent getting residual waste/salt on the outside of the column that can contaminate at a later time. Add 500 uL RPE to the column, and invert for ~2 mins before to eliminate salt from the inside of the column. Spin at 10,000 xg 1 min at room temperature.
• Repeat above for a total of two RPE washes
• If a lot of salt contamination, repeat wash step multiple times, increase volume of RPE to 700uL, and increase speed of centrifuge
• Wash column with 500 uL of 80% EtOH, spin 10,000 xg 1 minute at room temperature
• Transfer column to a new tube
• To dry tube, spin with tube lid open at 16,000 xg 5 mins at room temperature
• Any hint of EtOH will inhibit RNA elution
• Transfer column to new tube
• Add 14 uL of water to the column, close lid, incubate at room temperature for 1 min, and spin at 16,000 xg for 1 min
• Repeat elution step above with the 14uL flow-through.
• This can yield up to twice as much RNA

RNA isolation from 40,000 - 250,000 sorted T cells should yield 500 pg/uL – 11 ng/uL x 14 uL for gene expression profiling

Useful to optimize technique through inspection of RNA samples via NanoDrop for UV identification of contaminants and quantity estimate if have a high enough RNA concentration (Aranda et al., 2009), 2100 Bioanalyzer for quality of RNA and quantity estimate of low RNA concentrations, Qubit for specific quantitation of low amounts or RNA (5 ng in variable volume), and RT-PCR for identification of DNA