Figure 2
(A) Half-sclerotome dissection. Strips corresponding to the 10–18th most recently formed somites (S10-S18) were isolated from mouse embryos at TS15 (9.5–10.25 d.p.c.). Individual A- (green) or P-sclerotome (red) halves were dissected and pooled for further processing. V, ventral; D, dorsal; A, anterior; P, posterior. (B) Amplification of sclerotome RNA. (i) Total RNA was purified individually from pools of A- or P-half-sclerotomes. (ii) First strand cDNA was synthesized by reverse transcriptase (blue) using a modified Clontech SMART primer containing oligo (dT), a T7 RNA polymerase promoter and an universal primer (UP). (iii) The template-independent addition of 3' C residues allows second strand synthesis by strand-switching of reverse transcriptase using a second UP bearing G residues. (iv) Global cDNA amplification by limited PCR cycles using a UP alone. (v, vi) Further amplification, and generation of antisense-labelled probe by in vitro transcription using T7 polymerase (red) for hybridization to microarrays