Reverse Transcription/cDNA labelling and Hybridization

To each 5ug aliquot of mRNA, 1.5µl of 2µg/µl anchored dT primer (TTTTTTTTTTTTXN, where T=thymidine, X=any base except T, N=any base), and 3.5µl DEPC-ddH2O were added, and the tube heated at 70°C for 10 minutes, then placed on ice for 8 minutes. To each tube, 9.6µl Trimix (0.6µl 25mM dA,C,G/10mM dT, 0.6µl 5x Gibco RT buffer, 3µl 0.1M DTT, 5.4µl H2O), 4.4µl DEPC-ddH2O, 3µl Cy-3 or Cy-5 dUTP (or dCTP), 1µl RNAse inhibitor, and 2µl Gibco BRL Superscript II reverse transcriptase were added. In the germ line experiments, fem-3 and reference RNA were labeled with Cy-5, and fem-1 and wild type and glp-4 were labeled with Cy-3. The tubes were incubated at 42°C for 90 minutes. After cDNA synthesis, the RNA was degraded by adding 1.5µl of 1M NaOH and putting the tube at 65°C for 10 minutes, then the reaction was neutralized with 1.5µl of 1M HCl and diluted with TE to 500µl. The tubes were spun in Micron-30 (Amicon Bioseparations) for 8 minutes at setting 10 in a microcentrifuge, and the spin-through was discarded. An additional 500µl TE was added to the retentate, and respun through same column. The retentate in the column was then inverted, and collected in a fresh tube by spinning for 2 minutes at setting 10. The appropriate combinations of Cy-3 labelled cDNA and Cy-5 labelled cDNA were mixed until a purple color was achieved and add 1.5µl of 5µg/µl tRNA was added. This mixed sample was diluted again to 500ul with TE and spun through a new Micron-30 as before. The retentate was concentrated until a volume of about 10-12ul was achieved, and then collected by inversion and centrifugation into a fresh column. To this probe, 4µl 20x SSC, 0.5µl 10% SDS, and TE to a total volume of 27µl was added. The probe was heated at 100°C for 2 minutes, spun in at RT in a microfuge at full speed for 1-2 minutes. The probe was placed carefully on a coverslip, and then the microarray was lowered, DNA spot side down, until the coverslip adhered to the slide. The microarray was then placed in a waterproof hybridization chamber, and hydrated with a drop of 3xSSC on one end of the slide, away from the coverslip. The chamber was sealed, and the slide immersed in a 65°C waterbath for 20h.

 

Washes

After hybridization, the hybridization chamber was removed from the 65°C waterbath, dried off, and opened. The microarray slides were placed in glass slide racks, and washed in glass slide chambers as follows. The slides were immersed in 200 ml of 3x SSC/0.2% SDS and gently agitated until coverslip slid off, before dunking up and down for about 1 minute. The slides were then placed in a new slide rack, and immersed in 200 ml of 0.2x SSC and dunked up and down for about 1 minute. The slides in the same rack were then immersed in 200 ml of 0.1x SSC, and dunked up and down for about 1 minute. The slides were dried by centrifuging the glass rack in a table top centrifuge designed to hold 96-well plates for 3-4 minutes at setting 600-700 rpm. The slides were stored dry in a dark box until scanning.