Yeast Expression MicroArray Protocol

Feng Xu / MG, 1/17/2004, modified by Wei

I. Total RNA isolation (Qiagen RNeasy Midi Kit)

Day 1: Inoculate a 5 ml culture in YPD (or selective media) in the afternoon and grow overnight at 30 °C, shaking.

Day 2: Dilute into 50 ml YPD from starting OD600=0.2, grow at 30 °C to OD600=0.5

Enzymatic Lysis Protocol for Isolation of Total RNA from Yeast

Note: • Prepare Buffer Y1 Buffer Y1* 1 M sorbitol, 0.1 M EDTA, pH 7.4 Just before use, add: 1 ul of 14.3 M b-ME per 1 ml of Buffer Y1 100 U(1.43 mg 70 u/mg) lyticase/zymolase per 1 ml of Buffer Y1 • Use only freshly harvested cells for the enzymatic lysis protocol. • b- ME must be added to Buffer RLT before use. Add 10 ul b-ME per 1 ml Buffer RLT. • Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming to 37 °C and then place at room temperature.

1.Harvest yeast cells by centrifuging at 500 x g for 5 min at 4°C. Decant supernatant, wash once with 10 ml of cold H2O. (or asperate the remaining medium). Heat up centrifuge machine to 25C.

2. Resuspend cells in 2. 5 ml freshly prepared Buffer Y1 (1ml/108 cell) containing 35.7 mg lyticase (70 u/mg). Incubate for 30 min at 30°C. (or 2. 5mg zymolyase, 100u/ml buffer, make sure 2.5ul b-ME is added, 1ul/ml)

3. Centrifuge for 5 min at 500 x g (1500rpm) to pellet spheroplasts. Carefully remove and discard the supernatant.

4. Disrupt spheroplasts by adding 4 ml of Buffer RLT (make sure b-ME is added! 10ul/ml, total 40ul).

5. Homogenize yeast cells by vortexing the sample for 10 s, and pass the lysate 10 times through an 18–20-gauge needle fitted to an RNase-free syringe.

6. Centrifuge the yeast lysate for 5 min at 3000–5000 x g (4000-4300rpm for Js4.3 swinging bucket). Carefully transfer supernatant to a new 15 ml tube by pipetting.

7. Add 4.0 ml of 70% ethanol to the homogenized lysate, and mix thoroughly by shaking vigorously. (do not centrifuge!!)

8. Apply the sample to a RNeasy midi column placed in a 15 ml centrifuge tube (supplied). Maximum loading is 4 ml (load rest sample successively if more than 4ml) . Close the tube gently, and centrifuge for 5 min at 3000–5000 x g. Discard the flow-through.

9. Add 4 ml Buffer RW1 to the RNeasy column. Close the centrifuge tube gently, and centrifuge for 5 min at 3000–5000 x g to wash the column. Discard the flow-through.

10. Add 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and centrifuge for 2 min at 3000–5000 x g to wash the column. Discard the flow-through.

11. Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and centrifuge for 5 min at 3000–5000 x g to dry the RNeasy silica-gel membrane.

12. To elute, transfer RNeasy column to a new 15 ml collection tube (supplied). Pipet 200ul of RNase-free water directly onto the RNeasy silica-gelmembrane. Close the tube gently. Let it stand for 5 min, and then centrifuge for 3 min at 3000–5000 x g.

13. Repeat the elution step (step 12) as described with a second volume of RNase-free water.

14. Precipitated by ethanol: (Better use normal eppendorf tube rather than RNAse free tube since it might break under long time high speed centrifuge.

Measure elution volume (usually 340 ul, + 1020ul 100% ethanol +34ul NaAc (3M, pH=5.3) (1/10 volume), mix and put in –20C for 1 h, maximum speed spin 20min, wash with 75% cold ethanol. vacuum dried up and dissolved in 40ul RNAase free water (I put in 4C for 20min)

Dilute 1:1000, 0.5ul+500ul ddH2O to measure the OD, (I used Hall water, but dilute RNA immediately before transfer to measure cubicle)

15. Check RNA quality by agrose gel. 1% agrose gel. Be sure to use RNAZip to wash electrophoresis tank. Dilute 1ul RNA to 50ul, take 4ul mixed with 5ul loading buffer, 120V 40min

II. mRNA Purification (Qiagen Oligotex Maxi Kit) (optional, Feng and me didn’t use this actually. Go to RT directly) Oligotex mRNA Spin-Column Protocol For isolation of poly A+ mRNA from total RNA

Note: • Quantification of starting RNA Concentration of RNA sample = 40 ug/ml x A260 x dilution factor • Heat Oligotex Suspension to 37°C in a water bath. Mix by vortexing, and then place at room temperature. • Heat a heating block to 70°C, and heat Buffer OEB. • Buffer OBB may form a precipitate upon storage. If necessary, redissolve by warming at 37°C, and then place at room temperature.

1. Determine the amount of starting RNA. Pipet total RNA into an RNase-free 1.5 ml microcentrifuge tube, and adjust the volume with RNase-free water (if necessary) to the volume indicated in Table 4.

2. Add the appropriate volume of Buffer OBB and Oligotex Suspension (see Table 4). Mix the contents thoroughly by pipetting or flicking the tube. Spin-Column Protocol Table 4. Buffer amounts for Oligotex mRNA Spin-Column Protocol Total RNA Total RNA Volume Buffer OBB (ul) Oligotex Suspension (ul) Prep size ≤0.25 mg 250 ul 250 ul 15 Mini 0.25–0.50 mg 500 ul 500 ul 30 Midi 0.50–0.75 mg 500 ul 500 ul 45 Midi 0.75–1.00 mg 500 ul 500 ul 55 Midi 1.0–1.5 mg 650 ul 650 ul 85 Maxi 1.5–2.0 mg 650 ul 650 ul 115 Maxi 2.0–2.5 mg 650 ul 650 ul 135 Maxi 2.5–3.0 mg 650 ul 650 ul 175 Maxi

3. Incubate the sample for 3 min at 70°C in a heating block.

4. Remove sample from the heating block, and place at 20 to 30°C for 10 min. 5. Pellet the Oligotex:mRNA complex by centrifugation for 2 min at maximum speed (14,000–18,000 x g), and carefully remove the supernatant by pipetting. • Loss of the Oligotex resin can be avoided if approximately 50 ul of the supernatant is left in the microcentrifuge tube. The remaining solution will not affect the procedure.

6. Resuspend the Oligotex:mRNA pellet in 600 ul Buffer OW2 by vortexing or pipetting, and pipet onto a large spin column placed in a 1.5 ml microcentrifuge tube. Centrifuge for 1 min at maximum speed.

7. Transfer the spin column to a new RNase-free 1.5 ml microcentrifuge tube, and apply 600 ul Buffer OW2 to the column. Centrifuge for 1 min at maximum speed and discard the flow-through.

8. Transfer spin column to a new RNase-free 1.5 ml microcentrifuge tube. Pipet 20–100 ul hot (70°C) Buffer OEB onto the column, pipet up and down 3 or 4 times to resuspend the resin, incubate at 70°C for 1 min and centrifuge for 1 min at maximum speed.

9. Repeat the elution step (step 8) as described with a second volume of Buffer OEB. II. Reverse transcription All reagents are from Invitrogen unless noted

Oligo (dT) primer 2ul Total RNA 50 ug x ul RNase free water 9-x ul Total 11 ul

70 ∞C, 10 min, cool on ice for 3 min

add 5x first strand buffer 4 ul 0.1 M DTT 2 ul 25 mM dNTP mix 1 ul

Place tube at 37 ∞C for 2 min to pre-warm the mixture. Add 2 ul RTase, mix gently and incubate at 37 ∞C for 1 hour

Stop the reaction by adding 2 ul of 200 mM EDTA, mix. Add 2 ul of 1M NaOH to the reaction and incubate at 70 ∞C for 5-10 minutes Add 2 ul of 1 M HCl and mix well Add 1 ul of 1 M Tris pH 7.5 and mix well Purify cDNA by Qiagen PCR Purification Kit and elute cDNA in 30 ul EB buffer.

The following labeling and array hybridization steps are the same as acetylation array protocol (Please refer to Robyr et al., 2004, Methods.)