M. Fero. v.4

Materials

• RNA sample 30 µg Total or 2 µg mRNA
• Oligo dT (dT18VN) 5 µg/µL, 34.8 (µg/mL)/A260
• Superscript II, 5x 1st strand buffer (Invitrogen)
• 1 M DTT (Sigma, 43816, $16.80/10 mL)
• 50x dNTP Mix (dATP, dCTP, dGTP @25 mM, TTP=15mM)
• 10x AA-dUTP, 2 mM (MW = 523 g/mol), (Sigma A0410, $193.50/1mg - dissolve in 9.5 mL H2O)
• 1 N NaOH, 0.5 M EDTA, 2 M HEPES
• 0.2 M NaHCO3 (bicarbonate), 4 M NH2OH
• GFX spin columns
• QiaQuick columns
• PolyA (10 mg/mL) - Sterile filtered (0.45 µm)
• 20x SSC (175.5 mg/mL NaCl, 88.3 mg/mL NaCitrate)
• Cy3 and Cy5 Mono-reactive dye (Amersham PA23001 and PA25001):
  • To 1 tube dye add 36 µL anyhdrous DMSO and divide into 16x 2µL aliquots
  • Anhydrous DMSO: DMSO + Molecular Sieves (Sigma #M0133)

Notes

This procedure produces 1st strand cDNA with an anchored oligo dT primer and AA-UTP. It then couples couples amino reactive Cy-dyes to the AA-UTP. For spotted arrays control RNA should also be labeled using the opposing dye.

Procedure

A. 1st Strand Synthesis

1. In a 1.5 mL tube add:
   • RNA (30 µg total or 2 µg mRNA)
   • 1 µL (5 µg/µL) Oligo dT
   • q.s. 25 µL H2O
2. Incubate 70ºC, 10 min, then place on ice for 10 min.
3. Meanwhile, prepare RT Master Mix (per sample):
   • 2 µL Superscript II
   • 8 µL 5x 1st strand buffer
   • 0.4 µL 1 M DTT
   • 0.8 µL 50x dNTP mix
   • 4 µL 10x AA-dUTP (optional: spike with 1 µL 32P-dCTP)
4. Add 15 µL of RT Master Mix to each sample.
5. Incubate 42ºC, 2 hrs.

B. RNA Hydrolysis

1. To each sample add:
   • 12.5 1N NaOH
   • 5 µL 0.5 M EDTA
   • Incubate at 65ºC, 15 min.
   • Neutralize each sample with:
     • 20 µL 2M HEPES
     • O.K. to store o.n. at 4ºC.

C. Cleanup on GFX columns

1. Turn on Speed Vac refrigerators.
2. Add 0.5 mL GFX capture buffer. (optional: save 1 uL for 32P counts)
3. Load on GFX column. Spin 1 min. Discard flow-through.
4. Add 0.5 mL GFX wash buffer. Spin. Discard flow-through.
5. Elute in amber 1.5 mL tube:
   • Add 50 µL H2O. Incubate 1 min. Spin 1 min.
   • Repeat elution a 2nd time into the same tube. (Optional: save aliquot to measure 32P counts)
6. Dry in speed-vac.

D. Coupling to amino-reactive Cy dyes (e.g. Cy5 for sample, Cy3 for control)

1. Resuspend in 4.5 µL H2O
2. To a Cy-dye aliquot add: 2.25 µL 0.2 M NaHCO3
3. Quickly combine dye and DNA.
4. Incubate at r.t. in dark for 1 hr.
5. Quench by adding 4.5 µL 4 M NH2OH
6. Incubate at r.t. in dark for 15 min.

E. Cleanup in QiaQuick columns

1. Combine Cy5 and Cy3 samples (experiment and control).
2. Add: 70 µL H2O, 500 µL Buffer PB
3. Apply to a QiaQuick column. Spin 13K g for 30". Discard flow-through.
4. Add 750 µL Buffer PE. Spin. Discard flow through.
5. Repeat wash with PE.
6. Spin 1 min. to dry column.
7. Elute: Add 30 µL buffer EB, incubate 1 min. Spin 1 min. into a fresh amber tube.
8. Repeat elution 1x.
9. Speed-vac to dry down.

F. Preparation of Hybridization Solution

1. To each sample/control combination add:
   • 27.8 µL H2O
   • 5.4 µL 20x SSC
   • 2.8 µL polyA (10 mg/mL)
2. Load the DNA mixture on a 0.5 µm Millipore spin column
   • Spin at 12,000 g x5 min.
3. Store at -20ºC in the dark.

Tags: Fero-Lab-Protocols, Fero-RNA