Labeling involves the synthesis of double-stranded cDNA from total RNA followed by in vitro transcription (IVT). The quality of the T7-(T)24 primer is critical for the success of the whole experiment. It is essential that the primer is PAGE or HPLC purified, and we recommend verifying the quality of this primer before embarking on further experiments. The quality of the primer can be controlled for example by cDNA synthesis followed by IVT. During the IVT reaction, biotin-labeled cRNA transcripts are produced by a T7 RNA polymerase-catalyzed reaction in the presence of biotin-labeled CTP and UTP nucleotides. Use Qiagen RNeasy columns for purification of IVT samples. Do not use phenol/chloroform extraction to purify biotinylated samples. Compare also the Affymetrix white papers (9).

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