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
- 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
- Incubate 70ºC, 10 min, then place on ice for 10 min.
- 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)
- Add 15 µL of RT Master Mix to each sample.
- Incubate 42ºC, 2 hrs.
B. RNA Hydrolysis
- 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
- Turn on Speed Vac refrigerators.
- Add 0.5 mL GFX capture buffer. (optional: save 1 uL for 32P counts)
- Load on GFX column. Spin 1 min. Discard flow-through.
- Add 0.5 mL GFX wash buffer. Spin. Discard flow-through.
- 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)
- Dry in speed-vac.
D. Coupling to amino-reactive Cy dyes (e.g. Cy5 for sample, Cy3 for control)
- Resuspend in 4.5 µL H2O
- To a Cy-dye aliquot add: 2.25 µL 0.2 M NaHCO3
- Quickly combine dye and DNA.
- Incubate at r.t. in dark for 1 hr.
- Quench by adding 4.5 µL 4 M NH2OH
- Incubate at r.t. in dark for 15 min.
E. Cleanup in QiaQuick columns
- Combine Cy5 and Cy3 samples (experiment and control).
- Add: 70 µL H2O, 500 µL Buffer PB
- Apply to a QiaQuick column. Spin 13K g for 30". Discard flow-through.
- Add 750 µL Buffer PE. Spin. Discard flow through.
- Repeat wash with PE.
- Spin 1 min. to dry column.
- Elute: Add 30 µL buffer EB, incubate 1 min. Spin 1 min. into a fresh amber tube.
- Repeat elution 1x.
- Speed-vac to dry down.
F. Preparation of Hybridization Solution
- To each sample/control combination add:
- 27.8 µL H2O
- 5.4 µL 20x SSC
- 2.8 µL polyA (10 mg/mL)
- Load the DNA mixture on a 0.5 µm Millipore spin column
- Store at -20ºC in the dark.
Tags: Fero-Lab-Protocols, Fero-RNA