Microarray Center - Gene Expression Profiling

Technical support : Labeling 2/2

4. Procedure

4.1. Reverse transcriptionr

Combine the following reagents on ice :

Mix well and incubate at 70°C for 10 min. Snap-freeze on ice for 30 seconds, then centrifuge briefly and continue at room temperature.

Add the following :

Mix well and incubate at 42°C for 3 hours. Aminoallyl cDNA can be stored at -20°C.

Stop the reaction by adding 10 µL of 0.5 M EDTA, and then 10 µL of 1.0 M NaOH. Incubate at 65°C for 15 min to degrade RNA.

Add 60 µL of 1M Tris-HCl pH 7.4 to neutralise pH. Mix well (final volume = 110 µL).

Mix cDNAs with 550 µL (5x reaction volume) of buffer PB (Qiagen supplied), and transfer to a Qiagen column. Place the column on a 2 mL collection tube, and centrifuge at 13.000 rpm for 60 seconds.

Empty the collection tube, add 750 µL phosphate washing buffer (5mM KPO4 pH 8.5, 80% EtOH) to the column, and centrifuge at 13.000 rpm for 60 seconds. Repeat the procedure two additional times. After the last washing, empty the collection tube and centrifuge at maximum speed for 1 min.

Place the column on a fresh annotated collection tube (Ambion, cat#12480), carefully add 30 µL of phosphate elution buffer (4 mM KPO4 pH 8.5) to the center of the column membrane.

Incubate for 1 min at room temperature. Elute by centrifugation at 13.000 rpm for 60 seconds.

Again, add 30 µL of elution buffer. Incubate for 1 min, and elute by centrifugation. (final elution volume = 60 µL).

Add 20 µg (1 µL) of glycogen, 10 µL 3 M sodium acetate pH 5.2 and 200 µL 100 % Ethanol

Incubate at least 1 h at -80°C (prefably overnight).

At this point, aminoallyl-cDNA can be stored at -20°C. Take 1 µL aliquots for analysis by UV-spectrophotometry and 1% agarose-gel electrophoresis.

Note : The wash and elution buffers are substituted for the Qiagen supplied buffers because the Qiagen buffers contain free amines which compete with the ester-dyes during the coupling reaction.

Centrifuge precipitated cDNAs at 16.000xg for 20 min at 4°C. Wash the pellet with 750 µL of cold 75 % ethanol. (Do not invert the tube at any time. Collect instead the supernatant with a pipet).

Dry the pellet in a Speed-Vac at 30 °C for 5-10 min or until all liquid has been evaporated.

Ressuspend the pellet in 8 µL of freshly prepared 0.1 M carbonate buffer.

Dissolve one vial of reactive dye in 2 µL of DMSO (for one experiment, up to 6 µL/3 experiments have been successfully tested).

Vortex for about 10 secondes, and rapidly add 2 µL of the appropriate dye to the AA-cDNA. Vortex briefly to ensure that the reaction is well-mixed. DO NOT spin the tube, but instead, let the liquid settle by gravity.

To prevent photobleaching of the dyes, wrap the reaction tubes in foil and keep them away from light as much as possible.

Incubate the reaction for 1-2 hours at room temperature in the dark.

Notes :
1/ The dye esters are very sensitive to water, humidity and polluted air (ozone). The DMSO should be stored well-sealed in dessicant.
2/ Work as fast as possible, and treat each sample sequentially.
3/ Do not reuse the dyes since this will result in a lower coupling efficiency.

Add 80 µL of nuclease-free water and 10 µL of 3M sodium acetate pH 5.2 to the reaction mixture. Add 500 µL of PB buffer (Qiagen supplied). Mix well.

Transfer the cDNA mixture to a Qiaquick spin column placed in a 2 mL collection tube (Qiagen supplied), and centrifuge at 13.000 rpm for 60 seconds.

Empty the collection tube, add 750 µL of PE washing buffer (Qiagen supplied) to the column, and centrifuge at 13.000 rpm for 60 seconds. Repeat the procedure two additional times (3 washes in total).

After the last washing step, empty the collection tube and centrifuge 1 min at maximum speed to dry completely the column.

Place the column in a fresh annotated collection tube (Ambion), carefully add 30 µL of EB buffer (Qiagen supplied) to the center of the column, and incubate for 1 minute. Elute by centrifugation at 13.000 rpm for 60 seconds.

Elute a second time into the same tube with 30 µL of EB buffer (final volume = 60 µL).

Store labeled cDNA in the dark, and use within the day.

Take a 1-2 µL aliquot to check the labelling efficiency using the Nanodrop spectrophotometer.

For each sample, calculate the total amount of cDNA synthesised (picomoles) using :

pmol nucleotides = [A260 * volume (µL) * 37 ng/µL * 1000 pg/ng]/324.5 pg/pmol

Note : 1 A260= 37 ng/µL for cDNA; 324.5 pg/pmol average molecular mass of a dNTP.

For each sample, calculate the total amount of dye incorporation (pmol) using :

pmol Cy3 = A550 * volume (µL)/0.15

pmol Cy5 = A650 * volume (µL)/0.25

nucleotide/dye ratio = pmol cDNA/pmol Cy dye

Note :: >200 pmol of dye incorporation per sample and a ratio of less than 50 nucleotides/dye molecules is optimal for hybridizations.

<< | >>