Direct Labelling of Arabidopsis aRNA
1.
Prepare the following in an ice block. Place approximately 5µg of antisense RNA (aRNA)
(typically 2.5–10µg) into a sterile RNase-free 0.2ml PCR tube. Add 0.5µl of random nonamers
(3µg/µl) (Invitrogen) and 0.5µl of RNase OUT (Invitrogen). Add nuclease-free water to a final volume of 10.5µl.
2.
Incubate for 10 mins at 70 o C in a thermocycler. Place the samples in an ice block.
3.
Prepare a mastermix containing the following components (amounts given are for a single reaction):
4µl 5x SuperScript II First Strand Buffer (Invitrogen)
2µl 0.1M DTT (Invitrogen)
1µl dNTP mix (10mM dATP, 10mM dGTP, 10mM dTTP, 2mM dCTP)
1µl SuperScript II reverse transcriptase (Invitrogen)
4.
Add 8µl of mastermix to each RNA sample. Add 1.5µl of 25nmol Cy3- or Cy5- dCTP (GE Healthcare PA53021[Cy3-dCTP] PA55021 [Cy5-dCTP]) Mix well by gently pipetting (place samples in black box immediately after addition of dye. Centrifuge briefly. Incubate the samples at 42 o C for 2.5 hours. (NB: Cy dyes are extremely light sensitive.
After the addition of Cy dye all subsequent stages of the procedure should be performed in the
dark to minimise loss of fluorescence)
5.
Add 2µl of 2.5M NaOH to each of the labelled cDNA samples and incubate for
15 mins at 37 o C.
6.
Add 10µl of 2M MOPS buffer and place the samples on ice.
7.
Purify the labelled cDNA samples using Qiagen QiaQuick PCR Purification columns according to the manufacturer’s protocol. At the end of the procedure elute the purified sample with two 30µl aliquots of Buffer EB.
8.
Measure the concentration of purified labelled sample at 532nm (Cy3) or
635nm (Cy5) using the Nanodrop spectrophotometer. Take an aliquot of each sample equivalent to 40pmol (minimum of 30pmol) of Cy dye for subsequent array hybridisation. Alternatively, all of the purified labelled sample can be applied to the array.