QIAGEN OneStep Ahead RT-PCR Kit (200)

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Publication protocol

Translating ribosome affinity purification (TRAP). Tissue-specific TRAP was performed as described (Heiman et al., 2014). Briefly, 25 μl/sample Dynabeads Protein G (Invitrogen; 100- 07D) were collected on a magnetic rack and incubated with 10 μl anti-GFP antibody (Invitrogen A11122) for 2 hours at room temperature and then washed. More than 100 WT or miR-223-/-embryos injected with Tol2-tp63-GFP-L10a plasmid were collected at 1 hpw and homogenized by a prechilled Dounce homogenizer containing cell-lysis buffer on ice. Cell lysate supernatant was incubated with coated beads at 4 ̊C for 16-18h with end-over-end mixing on a tube rotator. After incubation, the beads were collected with a magnet and washed for four times with high- salt buffer. RNAs were eluted from beads and further purified by RNeasy MinElute kit (Qiagen). The quality of tissue-specific TRAP was identified by RT-PCR amplifying different cell type markers using OneStep Ahead RT-PCR kit (Qiagen). The quantification of the target genes was performed by one-step quantitative RT-PCR with SuperScript III Platinum one-step qRT-PCR kit (Invitrogen).

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