- Oligo-dT (T15) - Roche
- dNTPs (in buffer 50 mM Tris pH 8)
- RNasin - Promega
- Superscript II - Life Technologies
- Qiaquick PCR purification system - QIAGEN
- 22 x 50 mm cover slips
- Cot-1 DNA - Life Technologies
- Poly dA - Roche
- Cy3 and Cy5-dUTP
- 0.5M EDTA
- 2M NaOH
- 1M HCl
- 1M Tris pH 8
- 100mM sodium acetate
- 20x SSC
- 20% SDS
- Deionized Formamide
- MilliQ Water
- Array chip (5k gene-chip from QIMR)
- Hybridisation chamber
- Dnase-, RNase-free 0.5 and 1.5 ml eppendorf tubes
- Plastic slide holders
- 2 litre beakers
- Mix the following:
17 ul 25 ug total RNA or 1-5 ug of mRNA 1 ul RNasin 2 ul Oligo dT (2 ug/ul) Note: if the total volume of the RNA is > 17 ul add 1 ul of RNasin and speedivac down to 18 ul without heat.
- Heat to 70oC for 10 min. Cool on ice for 1 min.
- Prepare the following:
8 ul 5x RT buffer (supplied with Superscript II) 4 ul DTT (supplied with Superscript II) 1 ul dATP, dCTP, dGTP (each at 25mM) 2 ul dTTP (2.5mM) 2 ul Cy3 or Cy3-dUTP (1mM) - eg Cy3 for RNA from non-stimulated cells and Cy5 for RNA from stimulated cells
- Add the above mix to the RNA + oligo dT mix. Pulse spin and return to the ice. Add 1.5 ul of Superscript II. Incubated for 60 min at 42oC.
- Add another 1 ul of Superscript II and incubate for a further 30 min. Then quench the reaction on ice for 1 min.
- Add 1 ul of 0.5M EDTA and 2 ul of 2M NaOH. Heat to 65oC for 10 min. Hydrolysis destroys RNA.
- Add 4 ul 1M HCl and 4 ul 1M Tris pH 8.
- Add 17 ul of 100mM sodium acetate to each reaction.
Amended protocol for Qiaquick PCR purification kit
- Pool Cy3 and Cy5 reactions and add 425 ul PB buffer. Load the mixture onto column.
- Spin at 14K for 1 min. Discard eluate.
- Wash column with 650 ul of PE buffer.
- Spin at 14K for 1 min. Discard eluate.
- Spin at 14K for 1 min.
- Transfer column to fresh collection tube (1.5ml eppendorf)
- Add 40 ul of EB elution buffer to the column. Let stand 1-2 min. Elute purified cDNA by spinning at 14K for 1 min.
- To a fresh 0.5 ml tube add:
10 ul Cot-1 DNA (1 ug/ul) 2 ul poly dA (10 ug/ul) 40 ul purified cDNA probe
Speedivac down to a final volume of 11 ul.
- Add the following in order
8 ul 20x SSC 20 ul Deionized formamide 1 ul 20% SDS
Mix, spin down, and heat sample to 95oC for 5 min
- Incubate the labelled target at 45oC for 0.5-1.5 hr prior to placing on array. Temperature of probe is critical. Too hot leads to a scanning artefact, too cold and probe will precipitate. Make sure hybridisation chamber, slides and coverslips are ready before the next step.
- Place on ice for 30 sec, spin for 30 sec at room temperature.
- Place slide into hybridisation chamber. Add 10 of MilliQ water into the wells at each end of the chamber.
- Load cooled sample onto array slide and avoid bubles. Carefully place coverslip onto slide with fine forceps.
- Close and seal lid of hybridisation chamber and submerge in 45oC water bath for 14-24 hr.
- Prepare the following in 2 litres beakers
SDS Wash
990 ml MilliQ water 10 ml 20x SSC 2.5 ml 20% SDS
SSC Rinse
990 ml MilliQ water 10 ml 20x SSC - After hybridisation, dry chamber and undo lid. Using flat nosed forceps, hold array slide at frosted end (with coverslip still on it) and plunge into the SDS wash. Move slide sideways until coverslip falls off. Place slide into slide holder, and plunge holder for 15 sec, then rock for 3 min. Plunging every 30 sec or so.
- Rapidly transfer slide holder into SSC rinse, and plunge holder for 15 sec, then rock for 3 min. Plunging every 30 sec or so. SSC will start precipitating out if solution starts drying on slide.
- Remove slide with forceps and immediately spin slide at 500 rpm for 5 min at ambient temperature.
- Store slides in slide box with closed lid to reduce quenching of fluorophores, and scan Cy5 first, as it is more photolabile.
