Protocols:

•  Here is the protocol recommended by Affymetrix for standard Eukaryotic target labeling.


•  A suggested user-friendly version of the protocol for standard synthesis is also available.


•  However, since the standard procedure recommends to use 5 µg of total RNA as starting material for each target preparation reaction, a different protocol is necessary if much less RNA is available.
The small sample target labeling protocol utilizes two cycles of cDNA synthesis combined with in vitro transcription for target amplification (Eberwine et al., 1992).
The first cycle provides initial amplification of total RNA, resulting in unlabeled cRNA.
In the second cycle of IVT synthesis, biotin-ribonucleotides are incorporated to produce labeled antisense cRNA target