Chunk #41 — MATERIALS AND METHODS — RT-PCR and qPCR analysis

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Musculocontractural Ehlers-Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin.

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Total RNA was extracted with TRIzol® reagent (Ambion-Life Technologies). cDNA synthesis was performed using Reverse Transcriptase AMV (Roche) for whole embryos and SuperScript® II Reverse Transcriptase (Invitrogen Life Technologies) for CNC explants. RT-PCR was performed as previously reported (Hou et al., 2007). qPCR was performed on ten CNC explants per sample using the Fast SYBR® Green Master Mix (Ambion Life Technologies) and the StepOne™ Real-Time PCR System (Life Technologies). The relative expression was evaluated using the ΔCt method with the housekeeping gene eEF1A1 as the control. The primers are listed in Tables S1 and S2.