Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): III. Sample Purification.

CSH protocols

INTRODUCTIONT7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage. TLAD was designed primarily for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of dsDNA to generate microgram amounts of amplified RNA. Briefly, the strategy is to add a 3′ conserved end to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing, which permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step. IVT can then use this newly appended T7 promoter and linearly amplify the template dsDNA, producing antisense RNA (aRNA) product. After the IVT reaction is complete, the aRNA is cleaned up using the QIAGEN RNeasy Kit. This protocol for RNA sample purification is based on the manufacturer’s protocol for cleaning up RNA reactions, with minor modifications.