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Standard Operating Procedure

S.O.P#

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IGF0105.00

Title

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Fluorescent labelling of cDNA using the Stratagene Fairplay III microarray labelling kit

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1.0	BACKGROUND In order to identify differentially expressed genes, labelled cDNA targets are produced by first reverse transcribing the test and control total RNAs using modified nucleotides. The modification allows a secondary chemical coupling reaction to be carried out to attach a fluorescent label to the cDNA. These labelled cDNAs are then hybridised to probe genes on a microarray.
2.0	PURPOSE This procedure outlines the fluorescent labelling of total RNA using the Stratagene FairPlayÔ II microarray labelling kit.
3.0	RELATED SOPs Automated labelling of RNA using the Qiagen Biorobot 8000 SOP075.00
4.0	MATERIALS Amber tubes EQU#002 Stratagene FairplayÔII kit CS#003 3M Sodium Acetate CS#005 Cy3 monoreactive dye pack CS#006 Cy5 monoreactive dye pack CS#007 100% Ethanol CS#011 70% Ethanol RP#001 DyeEX spin kit EQU#053 1M sodium hydroxide RP#0028 1M hydrochloric acid RP#0029

5.0	cDNA PREPARATION During the labelling procedure, exposure to light should be kept to a minimum when using Cy dyes and mixtures containing Cy dye. At all times, keep reactions covered with foil. Before starting, prepare RNA at 2μg/μl by dilution or linear acrylamide precipitation (overnight).
	5.1				Set DriBlocks at 70°C and 48°C.
	5.2				Remove total RNA from -80°C freezer, and place on ice with, 500ng/ml oligonucleotide dT 10 x Stratascript reaction buffer 20 x dNTP mix 0.1M DTT RNase block (40U/ml)
	5.3				For each labelling reaction resuspend 20mg total RNA in 12ml of MilliQ water.
	5.4				Add 1ml of 500ng/ml oligonucleotide dT to the RNA, vortex and spin down. Incubate at 70°C for ten minutes.
	5.5				In an RNase free microfuge tube combine the following. For multiple labelling reactions make up a master mix containing enough reagent for the number of reactions you plan to carry out, plus a half reaction again to allow for pipetting errors. For each reaction add: 10 x Stratascript reaction buffer 2ml 20 x dNTP mix 1ml 0.1M DTT 1.5ml RNase block (40U/ml) 0.5ml Keep on ice until annealing incubation is finished.
	5.6				Cool the annealed primer for five to ten minutes at room temperature. Spin down before placing on ice until ready for use.
	5.7				Add 5ml of the master mix to the annealed primer and finally 3ml of StrataScript HC RT to each tube USING A P10 TIP TO IMPROVE ACCURACY. Vortex to mix, spin to return the contents of the tube to the bottom and place in a 48°C DriBlock for 60 minutes.
	5.8				Hydrolyse the RNA-DNA hybrid by addition of 10ml of 1M sodium hydroxide (Sigma) and incubate at 70°C for ten minutes. Cool to room temperature slowly (around 10 minutes). Do not cool on ice. Take glycogen for step 5.1 out of freezer to defrost.
	5.9				Spin the tube briefly to collect the reagents and add 10ml of 1M hydrochloric acid (Sigma) to neutralise the solution. Proceed directly to procedure 5.1.

6.0	cDNA PURIFICATION
	6.1	To the reaction, add in the following order (vortex after each addition); 4ml 3M sodium acetate 1ml 20mg/ml glycogen 100ml 100% ice cold Ethanol Incubate at -20 °C overnight The reaction can be stored at this point for up to two weeks.
	6.2	Spin the reaction using the Eppendorf microcentrifuge 5415D at 13000-14000 x g for fifteen minutes.
	6.3	Carefully, aspirate the supernatant and wash with 0.5ml 70% ice-cold ethanol. Return to the microcentrifuge and spin at 13000-14000 x g for fifteen minutes.
	6.4	Aspirate the supernatant and allow the pellet to air dry for approximately 10 – 15 minutes. Proceed immediately to procedure 6.1.
7.0	NHS-Ester containing dye coupling reaction
	7.1	Defrost coupling buffer in 37°C heating block to dissolve precipitate. Vortex to mix. Resuspend the cDNA pellet in 5ml of 2 x coupling buffer, vortex and heat gently at 37°C for 15 minutes to aid resuspension. (Remove Cy dyes for step 7.2 from freezer to defrost at this stage, make sure dye is at room temperature before use). Vortex and spin the cDNA plus coupling buffer after 15 minute incubation.
	7.2	A 10ml aliquot of Cy dye in DMSO is enough for two RNA samples. If necessary resuspend a fresh aliquot of Cy dye in 45 ml DMSO (supplied in the kit), and vortex gently to solubilise. Store dye in amber tubes -20 °C in the dark. These Cy dyes are stable for several months.
	7.3	Vortex and spin down Cy dye. Add 5ml of Cy dye to the cDNA and mix by gently pipetting up and down. Incubate for thirty minutes at room temperature in the dark. Keep tubes with Cy3 and Cy5 dyes in separate racks, eg Cy3 tubes in green rack and Cy5 tubes in pink rack. Proceed immediately to procedure 7.1.

8.0	DYE COUPLED cDNA PURIFICATION
	8.1	Take one Qiagen DyeEx spin column per reaction or one plate per 96 well plate and vortex to resuspend the resin.
	8.2	Loosen the cap of the spin column a quarter turn, snap off the bottom closure and place into a 2ml collection tube.
	8.3	Spin the columns using the Eppendorf microcentrifuge 5415D at 3000rpm for 3 minutes. Don’t tighten caps of columns completely.
	8.4	Carefully transfer the spin column to an amber tube and slowly apply the labelling reaction to the gel bed. Pipette the labelling reaction (~ 11μl) directly onto the centre of the slanted gel-bed surface. Do not allow the reaction mixture or the pipette tip to touch the sides of the columns. Pipette the sample slowly so that the drips are absorbed by the resin. Replace lid loosely.
	8.5	Return the tubes to the centrifuge, ensuring the column is placed in the same orientation in the centrifuge as previously. Spin the columns at 3000rpm for three minutes.
	8.6	Remove the spin column from the tube and discard. The eluate contains the purified labelling reaction.
	8.7	It may be advisable to pass an additional 10ul of MilliQ through the column and collect in a separate tube in case the label failed to elute from the column in the first pass.
	8.8	Keep column until QC gel has been run and checked. Store labelled cDNA at 4ºC in dark if using the same day. Otherwise, store at -20ºC for up to 4 weeks.

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