qRT-PCR for miRNA Expression Analysis

qRT-PCR was performed to determine the validity of miRNA by deep sequencing for expression profile analysis. Total RNA was extracted as described above. According to previous reports [20 (link), 39 (link)], the miRNA abundance was detected. Briefly, we first polyadenylated the total RNA (3 μg) including miRNAs, and then used the Mir-X^TM miRNA First-Strand synthesis kit (Clontech, Inc., Terra Bella, USA) to reverse transcribe poly (T) adapters into cDNA. Using the internal reference gene UBQ, we normalized the cDNA products [20 (link)]. These products were used as templates for qRT-PCR. qRT-PCR was performed on a LightCycler® 480 (Roche, Switzerland) using LightCycler 480 SYBR Green I (Roche, Switzerland). Along the entire miRNA sequence and adapter sequence provided by miRNA First-Strand synthesis kit (Clontech, Inc., Terra Bella, USA), the miRNA-specific forward primer for each miRNA and the universal reverse primer were designed. The primers were shown in the Additional file 1: Table S1. qPCR was performed according to the following protocol: 95 °C for 2 min, 40 cycles of 95 °C for 5 s and 60 °C for 20 s, followed by a thermal denaturing step to generate the melt curves. All of the reactions were performed in triplicate, including non-template controls. Statistical analysis was performed using the 2^-ΔΔct method [40 (link)].