2.6. Quantitative reverse transcription PCR (qRT-PCR)
First-strand complementary DNA was synthesized using 1.0 μg of total RNA
using oligo dT primer and Transcriptor Reverse Transcriptase (Penzburg,
Germany). qRT-PCR analysis was performed in LightCycler 480 SYBR Green I
Master mix (Penzburg, Germany) on a Roche 480 LightCycler® (Basel,
Switzerland). Complementary DNA (1:20 dilution) was used as a template
(5 μL) in a reaction volume of 20 μL. For each sample type, there were
four to six technical replicates. PCR cycles were as follows: initial
denaturation at 95 °C for 5 min, followed by 45 cycles of 95 °C for 10
s, 65 °C for 15 s, 72 °C for 12 s, and a final melt curve analysis to
determine the amplification of a single product. Primers were designed
using Primer-Blast
(http://www.ncbi.nlm.nih.gov/tools/Primer-Blast/)
to span an intron if possible, with 100-150 base pairs product size
(Supporting Information Table S2). MDP0000336547 was selected as
the reference gene (Bowen et al., 2014). Primer efficiencies and
relative expression were calculated using the Roche 480 Light Cycler
software.