Homogenise tissue in 300 µl TRIzol (Invitrogen) in a 1.5 ml tube using an RNAase-free polypropylene pellet pestle and add up to 700 µl of TRIzol. Centrifuge at 13,000 rpm in a microcentriuge for 10 minutes at 4 °C to pellet debris. Transfer supernatant to a fresh 1.5 ml tube. Add 0.2 volumes chloroform, shake vigorously for 15 seconds and incubate at room temperature for 2-3 minutes. Centrifuge at 13,000 rpm for 15 minutes at 4 °C. Transfer upper phase to a new RNase-free tube, being careful not to touch the interface. Add 0.7 volumes of isopropanol to precipitate the RNA. Incubate at room temperature for 5 minutes or 1 hour at -20 °C and then centrifuge at 13,000 rpm for 15 minutes at 4 °C. Discard the supernatant and wash the RNA pellet with 1 ml 70% ethanol/DEPC MilliQ water. Centrifuge at 13,000 rpm for 10 minute at 4 °C. Air-dry the pellet briefly (~ 5 minutes). Resuspend in an appropriate volume of DEPC MilliQ water, e.g. 20 to 50 µl. The RNA will dissolve more readily if the DEPC MilliQ water is preheated to 55 °C. Verify quality of RNA on a 1 % agarose gel and quantify with a Nanodrop.
Label
Cy3
Label protocol
To 3 to 5 µg of total RNA, add 1 µl 500 ng/ µl of Oligo(dT)23 anchored primer (Sigma) and adjust volume to 5 µl with DEPC- water. Incubate at 65 °C for 10 min, then snap cool on ice. Add 4 µl 5x First Strand Buffer (Invitrogen), 2 µl 0.1M DTT (Invitrogen), 1 µl 10 mM dNTP mix, 0.25 µl 40 U/µl RNasin (Promega) and 0.75 µl 200 U/µl Superscript III (Invitrogen). Incubate at 46 °C for 2 hours, then at 65 °C for 15 minutes, snap cool on ice. For second strand synthesis: Add 7.5 µl Second Strand Buffer (Invitrogen), 0.75 µl 10 mM dNTP mix, 2 µl 10 U/µl DNA Polymerase I (Invitrogen), 0.1 µl 5 U/µl RnaseH (Promega), 0.5 µl 10 U/µl E. coli Ligase (NEB) adjust volume to 40 µl with DEPC-water. Incubate at 16 °C for 2 hours, then purification using QIAquick PCR Purification Kit. To label take up to 1 µg double stranded DNA and make up to a total volume of 25 µl with DEPC-water. Add 20 µl 2.5x Random Primer Reaction Buffer (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 100 °C for 5 minutes, snap cool on ice. Add 1 µl 10 X low-C dNTP mix (5mM dATP,dGTP,dTTP, 2 mM dCTP), 2 µl 1 mM Cy3 or Cy5 dCTP (GE Healthcare) and 1 µl 40U/µl Klenow (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 37°C for 2 to 3 hours. Stop the reaction by adding 5 µl Stop Buffer (Invitrogen – BioPrime DNA Labelling Kit). Resuspend the resin in the AutoSeq G-50 column (GE Healthcare) by vortexing gently. Loosen the cap a quarter turn and snap off the bottom closure. Place the column in a collection tube. Pre-spin column at 5,000 rpm for 1 minute to remove the buffer. Remove the top cap and place column in a new 1.5 ml tube. Pipette half of the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Spin for 1 minute at 5,000 rpm. Discard the column. Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp. Reduce volume of probe to between 2 to 5 µl by placing in a speed vac with medium heat. Add 2 µl of 10 mg / ml sonicated salmon sperm DNA (Invitrogen).
Homogenise tissue in 300 µl TRIzol (Invitrogen) in a 1.5 ml tube using an RNAase-free polypropylene pellet pestle and add up to 700 µl of TRIzol. Centrifuge at 13,000 rpm in a microcentriuge for 10 minutes at 4 °C to pellet debris. Transfer supernatant to a fresh 1.5 ml tube. Add 0.2 volumes chloroform, shake vigorously for 15 seconds and incubate at room temperature for 2-3 minutes. Centrifuge at 13,000 rpm for 15 minutes at 4 °C. Transfer upper phase to a new RNase-free tube, being careful not to touch the interface. Add 0.7 volumes of isopropanol to precipitate the RNA. Incubate at room temperature for 5 minutes or 1 hour at -20 °C and then centrifuge at 13,000 rpm for 15 minutes at 4 °C. Discard the supernatant and wash the RNA pellet with 1 ml 70% ethanol/DEPC MilliQ water. Centrifuge at 13,000 rpm for 10 minute at 4 °C. Air-dry the pellet briefly (~ 5 minutes). Resuspend in an appropriate volume of DEPC MilliQ water, e.g. 20 to 50 µl. The RNA will dissolve more readily if the DEPC MilliQ water is preheated to 55 °C. Verify quality of RNA on a 1 % agarose gel and quantify with a Nanodrop.
Label
Cy5
Label protocol
To 3 to 5 µg of total RNA, add 1 µl 500 ng/ µl of Oligo(dT)23 anchored primer (Sigma) and adjust volume to 5 µl with DEPC- water. Incubate at 65 °C for 10 min, then snap cool on ice. Add 4 µl 5x First Strand Buffer (Invitrogen), 2 µl 0.1M DTT (Invitrogen), 1 µl 10 mM dNTP mix, 0.25 µl 40 U/µl RNasin (Promega) and 0.75 µl 200 U/µl Superscript III (Invitrogen). Incubate at 46 °C for 2 hours, then at 65 °C for 15 minutes, snap cool on ice. For second strand synthesis: Add 7.5 µl Second Strand Buffer (Invitrogen), 0.75 µl 10 mM dNTP mix, 2 µl 10 U/µl DNA Polymerase I (Invitrogen), 0.1 µl 5 U/µl RnaseH (Promega), 0.5 µl 10 U/µl E. coli Ligase (NEB) adjust volume to 40 µl with DEPC-water. Incubate at 16 °C for 2 hours, then purification using QIAquick PCR Purification Kit. To label take up to 1 µg double stranded DNA and make up to a total volume of 25 µl with DEPC-water. Add 20 µl 2.5x Random Primer Reaction Buffer (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 100 °C for 5 minutes, snap cool on ice. Add 1 µl 10 X low-C dNTP mix (5mM dATP,dGTP,dTTP, 2 mM dCTP), 2 µl 1 mM Cy3 or Cy5 dCTP (GE Healthcare) and 1 µl 40U/µl Klenow (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 37°C for 2 to 3 hours. Stop the reaction by adding 5 µl Stop Buffer (Invitrogen – BioPrime DNA Labelling Kit). Resuspend the resin in the AutoSeq G-50 column (GE Healthcare) by vortexing gently. Loosen the cap a quarter turn and snap off the bottom closure. Place the column in a collection tube. Pre-spin column at 5,000 rpm for 1 minute to remove the buffer. Remove the top cap and place column in a new 1.5 ml tube. Pipette half of the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Spin for 1 minute at 5,000 rpm. Discard the column. Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp. Reduce volume of probe to between 2 to 5 µl by placing in a speed vac with medium heat. Add 2 µl of 10 mg / ml sonicated salmon sperm DNA (Invitrogen).
Hybridization protocol
Add 140 µl of Ocimum hybridisation buffer to the labelled mixture and heat at 100 °C for 2 minutes on a hot-block. Centrifuge for 3 minutes at 13,000 rpm. Load 140 µl of the labelled sample to the microarray, and hybridise for 16 hours at 51 °C. Perform post-hybridisation washes as per PowerMatrix slides protocol (FMB). Pre-heat wash solution 1 (0.2 x SSC; 0.2% SDS) and wash solution 2 (0.2 x SSC) to 55 °C using a water bath. Incubate slides in a slide staining dish with pre-heated wash solution 1 on an orbital shaker at 50 rpm for 20 minutes. Transfer the slides to a staining trough containing pre-heated wash solution 2. Gently dip the slides up and down for 1 minute in wash solution 2. Repeat step in fresh wash solution 2 twice. Rinse the slides 3 times with fresh deionized water at room temperature (dip the rack in MilliQ water for 3 seconds). Transfer the slides to a clean microscope slide box with tissue at the base and centrifuge at 1000 rpm for 5 minutes. The slides are now ready to be scanned.
Scan protocol
Arrays are scanned at 5 µm resolution with a GenePix 4000B (Axon) dual laser scanner at 100% Power, with Lines to average = 1 and Focus position = 0 at their respective optimal PMT gains selected by the Auto-PMT function (0.005% saturation tolerance). Images are saved as Single-image TIFF.
Description
Abeta vs control, day 3 ab42-d3 vs 51D-d3, replicate 4 (dye-swap)
Data processing
The raw data were filtered to remove any probes that were rejected in over 50% of samples and were quantile-normalised across all arrays using Limma. Missing values were imputed using the impute package for R
