Validation of miRNA expression using qRT-PCR

Poly (A)-tailed RT-qPCR was used to measure the gene expression variation and validate the deep sequencing results of miRNAs. Reverse-transcription reaction and real-time PCR primer design were conducted according to previous report (Shi and Chiang, 2005) and the manufacturer's instructions (Takara-Primescript miRNA qPCR starter kit, 2.0), where each PCR reaction was performed in a volume of 25 μl containing 12.5 μl of SYBR Premix Ex Taq II (2×), 1 μl of each forward primer, 1 of μl universal reverse primer and reverse-transcribed cDNA from ~100 pg of total RNA. The PCR protocol was 5 s at 95°C, 40 cycles of 95°C for 5 s, 60°C for 20 s and 72°C for 1 min. Real- time PCR was performed on the Applied Biosystem 7500 Fast detection system using the SYBR Green I method, and all the reactions were run in biological triplicates. The melting curve was used to determine the specificity of PCR products (primer amplicons).

The delta-C_T (corresponding cycle threshold) method was used to calculate the relative expressional levels of miRNAs (Livak and Schmittgen, 2001). In this method, the C_T values of the wanted miRNAs and reference genes were first transformed for measurement using delta-C_T followed by dividing the quantities of wanted miRNAs by the geometric mean of the reference genes. The standard deviation and mean values were calculated using to triplicates RT-qPCR assays. The C_T was calculated using the machine accessory software and converted into relative copy numbers using a standard curve as previously described reports (Jones-Rhoades and Bartel, 2004; Shi and Chiang, 2005). The gene 5.8 S rRNA was used as reference gene in the qPCR detection of miRNAs. Student's t-test was used for the statistical analysis of the RT-qPCR.