These methods are basically an implementation of the Protocols recommended by Affymetrix, with small modifications. Essentially, a large amount of total RNA is harvested from cells (G1E cells are presumed below), purified, and biotinylated cRNA is generated. This cRNA is chemically fragmented and then hybridized to the GeneChip. Reagents used in this procedure and the GeneChips themselves are expensive, so be careful. The steps involved are:
Harvest Log Phase Cells
Dilute a log-phase culture such that experiment begins with 40-45 x 106 cells per condition tested. These will end up as T-150s with 100 mL of 0.5 x 106/mL.
Pool cells into a T-225, make appropriate dilution, then aliquot out into T-150s, this assures that cultures have a common starting point and initial conditions. If possible, remove cells to remain untreated and add the differentiation agent and then aliquot. Again, this reduces experimental error.
At the appropriate time, spin cells down at 1200 rpm (RCF ~ 300g) for five minutes at 4C.
Resuspend in 10 mL PBS, combine into a single 15 mL conical tube.
Spin again to pellet.
Resuspend in 1 mL PBS. Place immediately on dry ice and freeze solid. When convenient, transfer to -80C freezer.
Recovery of mRNA
Trizol or STAT-60 Reagent = phenol + guanethidium
Add 1 mL of Trizol per 10 x 106 cells. Typically, 5-9 mL. Titurate with pipette to dissolve. Vortex. Shear with a syringe and needle if necessary, but get it into a clear solution.
Allow to stand at room temperature for five minutes.
Add 0.2 mL of chloroform per 1 mL of Trizol. Shake vigorously for 15 seconds. Be sure that chloroform does not smear the labels on tubes. Allow to stand at room temperature for 2-3 minutes.
Centrifuge at 3400 RPM in Beckman, or 13,000 RPM in desktop centrifuge for 15 minutes at 4C. This is to separate phases.
Transfer the upper, clear aqueous phase with the RNA to another tube; avoid the interface. Volume should be about 60% of the volume of Trizol used.
Precipitate RNA by adding 0.5 mL of room temperature isopropanol per 1 mL Trizol. Allow to stand 5 minutes at room temperature. Ideally, there is a separate supply of isopropanol available for RNA work.
Centrifuge at 3400 RPM in Beckman or 13,000 rpm in desktop centrifuge at 4C for 10 minutes.
Carefully aspirate the supernatant.
Allow the RNA pellet to air dry briefly (but not so much as to become insoluble).
Resuspend in 0.5 mL of DEPC
Add 50 uL 5M NaCl
Add 1 mL 100% EtOH
Store at -80C overnight to precipitate.
Spine at 13,000 rpm in desktop centrifuge at 4C for 15 minutes to pellet.
Wash once with cold 80% ethanol.
Centrifuge at 3400 rpm in Beckman or 13,000 rpm in desktop centrifuge at 4C for 5 minutes.
Aspirate supernatant. Briefly air dry. Resuspend in 200-300 uL DEPC H2O.
Check optical density of samples
Combine 2 uL sample with 300 uL H2O, mix well.
Turn on spectrophotometer. Select 260 nm. Zero on H2O blank.
RNA is 40 ug/mL per OD unit.
Check RNA quality
May run 8-15 ug of RNA per lane on a 1.2% agarose gel with formaldehyde. Ethidium in loading buffer permits visualization of the 18S and 28S RNAs. The RNA from this gel may be probed per Northern procedure for known transcripts to confirm induction of message.
RNA purification with Rneasy Columns
Rneasy columns are rated at 100 ug, but tolerate 125 ug.
If RLT buffer has precipitated, warm it. Prepare enough RLT for all samples. Before using, mix 10 uL beta-mercaptoethanol per mL of RLT. Add 350 uL of RLT per 100 uL RNA solution. Mix well by pipetting.
Add 250 uL of 100% ethanol (per 100 uL of RNA solution).
Apply the sample to an Rneasy spin column. Centrifuge at 10,000 rpm for 15 seconds. If necessary, keep applying sample and spin it through the column. RNA will be retained. Discard flow-through.
Transfer the column to a new collection tube. Add 500 uL RPE and centrifuge for 15 seconds at 10,000 RPM. Discard flow-through.
Rince again with another 500 uL RPE. Spin briefly to collect bulk of fluid, then replace tube and spin an additional 2 minutes to dry.
Transfer the column to a new collection tube. Add 30-50 uL of Rnase free water directly onto the membrane. Spin at 10,000 rpm for one minute to elute.
Check OD of eluant, run on gel to check purity.
cDNA Synthesis: First Strand
Refer to Affymetrix GeneChip Manual, Chap 2, Sec 4
Use 24 ug of double-purified RNA per reaction, adjust to 9 uL with DEPC H2O.
Combine 9 uL of RNA with 1 uL of T7-(dT)24 primer.
Vortex and quick spin.
Denature at 70C for 10 minutes. Quick spin. On ice.
Prepare slight excess of reaction buffer for all reactions.
On ice, add 10 uL of reaction mixture per sample.
Incubate at 42C for one hour.
Place on ice. Quick spin.
|5X Buffer||4 uL|
|0.1M DTT||2 uL|
|10 mM dNTPs||1 uL|
|Superscript II RT||3 uL|
cDNA Synthesis: Second Strand
Prepare slight excess of reaction buffer for second strand synthesis
Combine above mixture.
Flick to mix. Quick spin. Incubate for two hours at 16C (large styrofoam box is okay, use a floating rack). Quick spin.
Blunt overhangs by adding 2 uL (=10 U) T4 DNA polymerase. Mix. Quick spin.
Incubate 5 minutes at 16C.
Add 10 uL 0.5M EDTA. May store at -20C.
|DEPC H2O + first strand rxn||91 uL|
|5X 2nd strand buffer||30 uL|
|10 mM dNTPs||3 uL|
|E.coli 10 units/uL DNA ligase||1 uL|
|E.coli 10 units/uL DNA pol||4 uL|
|E.coli 2 units/uL RNase H||1 uL|
Pellet phase-lock gels at 13,000 rpm for 20 seconds.
Add 162 uL PCI (equal to final reaction volume). Vortex.
Transfer to PLG tubes (no vortex).
Spin at 13,000 rpm x 2 minutes.
Transfer upper phase to 1.5 mL Eppendorf
Add half volume (i.e., 81 uL) 7.5 M NH4Ac
Add 610 uL of cold 100% EtOH (store at -80C)
Spin 13,000 rpm x 20 minutes at room temperature
Add 50 uL DEPC H2O
Add 350 uL 100% EtOH
Decant (if pellet loose, spin again)
Wash with 1 mL of cold 80% EtOH.
Air dry briefly
Resuspend pellet in 12 uL DEPC H2O
(if stopping here, freeze at -80C)
IVT of Biotinylated cRNA
Prepare "BioArray High Yield IVT" transcription buffer.
Add 37 uL of the reaction mixture to 3 uL of each cDNA from above; total 40 uL.
Mix, quick spin, incubate at 42C for 4 hours. Gently mix every 30 minutes.
(if stopping here, can quick spin and place on dry ice)
|DEPC H2O||19 uL|
|10X HY Buffer||4 uL|
|10X dNTPs||4 uL (with biotinylated dCTP and dUTP)|
|10X DTT||4 uL|
|10X RNase Inhibitor mix||4 uL|
|20X T7 RNA polymerase||2 uL|
Purification of IVT transcripts
Expect about 50-100 ug of biotinylated RNA per reaction
add 60 uL DEPC H2O per above reaction, final volume 100 uL
Purify on RNeasy column as previously
biotinylated RNA will not partition correctly with PCI
Need to get rid of unicorporated dNTPs to later quantitate cRNA spectrophotometrically.
Wait a minute, and then elute in 40 uL DEPC H2O
Check OD to determine yield. A260/A280 should be 1.9-2.1
Fragmentation of Biotinylated target RNAs
Heat at 94uC x 35 minutes, then place on ice.
Run about 2 uL of each sample (about a microgram) on a 1% agarose non-denaturing gel. Run 100 bp and 1000 bp ladders for size estimation.
|16 ug of biotinylated cRNA + DEPC H2O to a total volume of 24 uL|
|6 uL 5X fragmentation buffer|