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HYBRIDIZATION & BINDING ASSAYS

HYBRIDIZATION ASSAYS ON DNA ARRAYS

We have selected Dendrimer system from Genisphere for probe labeling and signal amplification of sample RNA for ease of application with excellent signal to noise ratio. Hybridization procedure adopted by KamTek for glass slide microarrays involves the following steps:

Preparation of Total RNA Sample

We request our customers to provide at least 10ug of good quality mammalian total RNA and 20ug of plant total RNA in no more than 20ul of RNase free water. The quality of RNA is determined from the gel electrophoresis pictures and the A260/A280 ratio also required to be submitted with the RNA sample. We recommend the use of Qiagens RNeasy kits for RNA isolation. RNA samples should be shipped to KamTek on dry ice.

cDNA Synthesis

We have standardized our protocols for use with the 3DNA Dendrimer detection/signal amplification system where a capture sequence linked to either oligo-d (T) or random primer is incorporated at the end of cDNA.Standard RT reaction is used for this purpose. Preparation of cDNA is purified and concentrated using Millipore Microcon filter columns. Since fluorescent dyes are not used in the RT step, the procedure is free from any bias due to unequal incorporation of dyes, and also does not require handling of reagents and performance of reaction in dark.

Post Processing of Microarrays

The microarrays prepared at KamTek are post processed and denatured by our Standard Operating Procedures. One of the arrays per printing batch of 20 slides is stained with SYBR Green to confirm the quality of spots.

Hybridization of cDNA to Probe DNA on the Microarrays

Concentrated preparations of cDNA with capture sequence tails (different sequences for Cy3 and Cy5 dendrimers) are hybridized to already processed and denatured microarrays on slides using optimized hybridization conditions. Hybridization mixture containing cDNA is placed between the slide and coverslip and incubated in a moist chamber at a predefined temperature. Hybridization buffer used with the system can be with or without formamide depending on the preference of the investigators. The hybridized arrays are given stringent washes with ultra-clean SSC wash buffers and dried for the next step. Again this step does not require to be performed in dark since no light sensitive reagents are involved in this step.

Detection of Hybridization Signal

The anchored cDNAs are detected by a second hybridization with fluorescent-labeled DNA Dendrimers carrying oligomer sequences complementary to the capture tail sequences on the cDNA molecules. Experimental and control Dendrimers are labeled with different fluorophores each with its own specific capture tail sequence. Hybridization is carried out in hybridization buffer with or without formamide and at the same temperature as used in the previous step. The hybridized arrays are given stringent washes with ultra-clean SSC wash buffers, and dried and scanned immediately thereafter. All of the processes under this step are conducted in dark.

Binding Assays on Protein and Glycoarrays

A wide variety of binding assays can be performed on these arrays, each adapted to the array being used in the assay procedure and preferred signal molecule to be used. On the glass slides, fluorescent tag conjugated to an antibody or streptavidin molecule is a preferred choice with our scanning system. We provide recommendations to our clients based on their specific needs.
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