ethanol precipitation. The RNA was vacuum dried and resuspended in RNase-free water to a final concentration of 1 g/l.

Target labelling reaction

Labelling was performed using a modified version of the published protocol from the DeRisi laboratory (www.microarrays.org). The modifications included an increase in the ratio of amino-allyl-dUTP:dTTP to 6:4. The neutralisation was performed with Tris-HCl. All reverse transcription reactions were performed at 42C using 15 g of total RNA previously extracted as described above. The total unit amount of Transcriptor Reverse Transcriptase was 20 units per reaction (10 units added, and 1 hour later another 10 units were added). The total unit amount of product Y was 400 units per reaction (400 units added and left for 2 hours according to the manufacturers recommended protocol). The labelled product was vacuum dried and finally resuspended in 34.5 l HGMP hybridisation buffer (40% formamide, 5x Denhardts solution, 5x SSC, 0.05 M Tris-HCl pH 7.4, 1.0 mM sodium pyrophosphate, 0.1% SDS), 0.4 g yeast tRNA, 0.96 g poly-dA and 2.7% SDS (www.hgmp.mrc.ac.uk/Research/Microarray/ index.jsp).

Hybridisation

The microarrays used in this study are from a single printing run, custom made with approximately 12,000 sequenced expressed-sequence tags (ESTs) from a nonnormalised library. The ESTs were PCR amplified and the products used for printing were analysed for content and quality on agarose gels. Every microarray was visually inspected and samples were prescanned for printing defects. The microarrays were preblocked (10% BSA, 3x SSC) for 1 hour at 55C. Slides were washed with Milli-Q water (Millipore), and dried by centrifugation. Fixation of cDNA products was achieved after boiling the product and submersing
'"/>

Source:


Page: All 1 2 3 4 5 6 7
Related biology technology :

1. Transcriptor Reverse Transcriptase
2. Increase the Power and Sensitivity for Your cDNA Synthesis with the New Transcriptor Reverse Transcriptase
3. Properties of Reverse Transcriptases
4. Reverse transcriptases suitable for one-step RT-PCRb
5. Reverse transcriptases suitable for two-step RT-PCRd
6. C. therm. Polymerase for Reverse Transcription in Two-Step RT-PCR
7. Recombinant HIV Reverse Transcriptase
8. Novel Bonding Chemistry Imparts Enhanced Polar Selectivity to TSK-GEL ODS-100V Reversed Phase Columns
9. New Microarray Labeling Kit for Preparing cDNA Probes
10. Stratagenes Human cDNA Microarrays: Perform Gene Expression Profiling and Discover New Genes
11. An Optimal DNA Microarray Substrate for the Identification of Fetal and Somatic Liver Stem Cells of the Rat