Protocol 53 Hybridization washing and scanning

For every co-hybridization two RNAs were labeled in a reverse transcriptase (RT) reaction, RNA from undifferentiated and from differentiated cells, resulting in three hybridizations each for the BDNF series and the NT4 series (Figure 5.1). To account for dye-specific effects every hybridization was done twice, with the dyes exchanged by flipping the RT primers so that every RNA/cDNA was labeled once with the Cy3-specific capture sequence and once with the Cy5-specific sequence. Thus, six arrays were used for each series (NT4 and BDNF), adding up to 12 hybridizations in total.

Before hybridization, the spotted material was rehydrated by holding the slides over hot water until a vapor coating appeared, and quickly dried by placing them on a hot plate (98°C) for 3-5 s. Then the spotted material was crosslinked with the slide's surface by two successive UV crosslinking steps (120 mJ) in a UV Stratalinker 1800 (Stratagene, Amsterdam, The Netherlands). Remaining chemically active sites on the surface were blocked by 15 min incubation in succinic anhydride/sodium borate solution. Afterwards, the slides were briefly washed in ultrapure water and dried by centrifugation (125 g, 3 min, room temperature) and 3-5 s incubation on the hot plate (98°C). They were now ready for hybridization.

Labeling and hybridization reactions were performed using the 3DNA Array 50 Expression Array Detection Kit (Genisphere, Hatfield, PA; Figure 5.3). The labeling kit was used according to manufacturer's instructions. For each labeling reaction, 20 ^g of total RNA was deployed. In brief, RNA and RT primers were mixed, heat denatured, and RNase inhibitor was added. Then a reaction mix consisting of the Superscript II RT (Invitrogen), Superscript II reaction buffer, dNTPs, and DTT was pooled with the RNA/RT primer mix. The RT was allowed to react for 2 h at 42°C before the reaction was stopped and the DNA/RNA hybrids were heat denatured.

The primers for the reverse-transcription contained a poly-T sequence which bound to the poly-A tail of the mRNA. In addition they included a capture sequence which allowed for discrimination of the two cDNA pools after hybridization of synthesized cDNA to the array.

After cDNA synthesis and incorporation of the capture sequences, the cDNA was concentrated according to the manufacturer's instructions. Briefly, linear acrylamide, NaCl, and ethanol were added to the reactions. The mix was incubated at -20°C for 30 min and then centrifuged at 12,200 g at room temperature for 15 min. After aspiration of the supernatant the pellets were washed with 70% ethanol, recentrifuged for 5 min, and dried in a heat block at 65°C for 20 min.

For cDNA hybridization to the array the pellets were carefully resuspended in 10 ^l of nuclease-free water and heated to 65°C for 10 min. Then the final hybridization mix was prepared and incubated at 75°C for 10 min and 45°C for 20 min. Meanwhile the array was prewarmed to 45°C for 15 min. The final hybridization mix (cDNA) was mixed, centrifuged briefly, applied to the prewarmed array and covered with a coverslip. The slide was put into a sealed humidified chamber and was incubated in a water bath at 42°C overnight.

The next day the 3DNA capture reagents, that is the dendrimers, were hybridized to the array. First, the array was washed by sequential incubation in 2 x SSC/0.2% SDS for 10 min, in 2 x SSC for 10 min, and in 0.2 x SSC for 10 min at room temperature. To remove remaining liquid from

Reverse transcribe with unlabeled dATP, dTTP, dGTP and dCTP


RT Primer Oligo






Stop RT reaction and degrade RNA

cDNA hybridization to Microarray

Wash away unbound cDNA, then add 3DNA capture reagent to bind to cDNA on Microarray

Wash and scan Microarray cDNA

-3DNA Capture Sequence

Microarray i I

3DNA Capture Reagent Containing ~45 fluorescent dyes per molecule complement to cDNA capture sequence complement to cDNA capture sequence

the slide surface, it was transferred into a slide holder and centrifuged (125 g, 3 min). Meanwhile the 3DNA capture reagents were thawed in the dark for 20 min. 3DNA capture reagents and the hybridization buffer were then heated to 55°C for 10 min, and an anti-fade reagent was added to the hybridization buffer. The final hybridization mix (3DNA) was prepared, mixed very carefully, and incubated at 75°C for 10 min followed by an incubation at 45°C for 20 min. The array was again prewarmed as before. The mix was applied to the array and the slides were kept in a dark humidified chamber at 42°C for 3 h. Subsequently, the array was washed as before in 2 x SSC/0.2% SDS for 10 min, 2 x SSC for 10 min, 0.2 x SSC for 10 min, and finally briefly in deionized water. The slide was dried by centrifugation (125 g, 3 min) followed by an incubation at 42°C for 5 min. It was stored in a dry and dark box until scanning.

The arrays were scanned with the Affymetrix 428 Array Scanner (Affymetrix, Santa Clara, USA). Fluorescence intensities of Cy3 and Cy5 were measured separately at 532 nm and 635 nm. The photomultiplier tube gain was typically between 40-55 dB for Cy3 and 35-45 dB for Cy5. This ensured that the signal intensity reached saturation in less than 1% of the spots. The resulting images were saved as 16-bit data files in tag image file format (TIFF).

Table 5.2

Final hybridization mix (cDNA) Final hybridization mix (3DNA)

Concentrated cDNA 10 |l Hybridization buffer + Anti-Fade 25.0 |l

Hybridization buffer 22 |l 3DNA capture reagent 1 (Cy3) 2.5 |l

Array 50 dT Blocker 2 |l 3DNA capture reagent 2 (Cy5) 2.5 |l

Cot-1 DNA 2 |l Nuclease-free water 5.0 |l

Figure 5.3.

Labeling and detection of mRNA with Genisphere dendrimer technology. (A) Messenger RNA is reverse transcribed into cDNA using unlabeled nucleotides and a poly-T oligo, that carries a capture sequence. Two such cDNA populations are co-hybridized to the microarray. These are specifically detected by the 3DNA capture reagent. (B) The capture reagent contains dendrimers, large molecules composed of DNA strands coupled to fluorescent labels (Cy3 and Cy5, depicted as circles). The dendrimers present sequences complementary to the capture sequences on the cDNA, thereby allowing the detection of cDNAs derived from one RNA pool only. In consequence, a single cDNA molecule attracts approximately 45 fluorescent labels (Cy3 or Cy5) to the microarray, leading to a higher sensitivity of this method compared to the hybridization of cDNA directly labeled during reverse transcription with Cy3- or Cy5-coupled nucleotides.

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