Once double-stranded cDNA was synthesized, the DNA was labeled using a NimbleGen One-Color labeling kit (Roche NimbleGen, Inc., Madison, WI, USA). The samples were then labeled with Cy3-random nonamers (supplied in the kit). Based on the concentrations (determined using a NanoDrop ND-1000; Thermo Fisher Scientific, Inc.) of the labeled products, the volume of the Cy3-labeled sample in each tube was calculated (1 µg cDNA in 40 µl diluted Cy3-random nonamers). Equal quantities of samples were used for subsequent stages of the hybridization experiments.
Nimblegen one color labeling kit
Manufactured by Roche
Sourced in United States
The NimbleGen One-Color labeling kit is a tool used for labeling DNA or RNA samples in preparation for microarray analysis. The kit provides the necessary reagents and protocols to label samples with a single fluorescent dye, allowing for the quantification and comparison of gene expression levels across different samples.
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2 protocols using nimblegen one color labeling kit
1
Fluorescent Labeling of cDNA Samples
2
Microarray Profiling of Genomic DNA
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Genomic DNA (gDNA) was isolated from frozen and FFPE samples as described above. The gDNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies). Then, 1 μg gDNA was fluorescently labeled with Cy3-labeled random nonamers using the NimbleGen One-Color Labeling Kit (Roche). Labeled gDNA was re-quantitated with the Qubit 2.0 Fluorometer and 2 μg of the labeled gDNA was hybridized to the 12×135K format of the LLMDA v.5 for 60 hr. The arrays were washed using a NimbleGen Wash Kit (Roche) and scanned at 532 nm (Cy3 channel) using the NimbleGen MS 200 Microarray Scanner (Roche). The array was analyzed using the Composite Likelihood Maximization method at the 99% threshold (signal intensity of target probes were 99% above random control probes) [17 (link),25 (link)].
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