The Covance Genomics Laboratory (CGL) offers several amplification protocols to meet our collaborators needs. Contact us to determine which amplification protocol will best meet the needs of your project.

Amplification Options	Standard Input	Low Input	Ultra Low Input
Affymetrix
Transcript Representation Amplification Protocol Target Material Label Preferred Experimental Design	3' bias Ribo-SPIA cDNA Biotin end labeling Intensity	3' bias Ribo-SPIA cDNA Biotin end labeling Intensity	3' bias 2xRT/IVT cRNA Biotin-UTP incorporation Intensity
Amplification Options	Standard Input	Low Input	Ultra Low Input
Agilent
Transcript Representation Amplification Protocol Target Material Label Preferred Experimental Design	3' bias RT/IVT cRNA Aminoaly I-UTP incorp Ratio	3' bias 2xRT/IVT cRNA Aminoally I-UTP incorp Ratio *Preferred protocol for FFPE profiling	3' bias Modified 2xRT/IVT cRNA Aminoally I-UTP incorp Ratio

Click on an amplification protocol above for more information.

Amplification Process Overview
CGL's sample amplification and labeling protocol is a customized system utilizing automated liquid handling. Samples are processed in parallel in 96-well plates to minimize potential variation. Reaction purification is achieved using magnetic binding beads for cDNA and Qiagen RNeasy kits for cRNA purification. The final in vitro transcription incorporates biotin moieties that are later labeled with phycoerythrin.

Pooling and Splitting
Although possible, pooling samples after amplification is rarely required or advised. Due to the stability of the platform and the flexibility of the single channel intensity data, biological replicates are more beneficial than technical or hybridization replicates.

Labeling
The Affymetrix GeneChip system utilizes a single dye channel for detection. Biotin is incorporated during the amplification. This binds a streptavidin-conjugated phycoerythrin that provides the measurable signal.