Quantitative real-time PCR
We selected 19 (skin:10, brain:9) unigenes for sequencing results
validation by qRT-PCR. The cDNA was transcribed from 2μg of total RNA
using the Prime- ScriptTMII 1 st Strand cDNA Synthesis Kit in 20 μL of
reaction mixture. qRT-PCR was performed with the Roche
LightCycler® 96 Detection System (Applied Biosystems,
Foster City, CA, USA) with Roche LightCycler®96 SYBR
Green I Master (CWBIO, Beijing, China).The thermal profile for SYBR
Green I RT-PCR was 95 °C for 5 min followed by 40 cycles of 95 °C for 10
s, 60 °C for 10 s and 72 °C for 10 s. Gene ACTIN was used for
normalization. The 2−ΔΔCT method was used to analyze
the relative expression of these genes. All reactions were carried out
in triplicate. Primers are listed in
(Table .1.A; Table .1.B).