Mircury lna universal rt microrna pcr primer sets

For miRNA expression reverse transcription (RT) was carried out with the Universal cDNA Synthesis kit from Exiqon using 20 ng of total RNA. qPCR was performed using the ExiLENT SYBR Green master mix and miRCURY LNA Universal RT microRNA PCR primer sets following manufacturer’s recommendations (Exiqon INC., USA).
For target gene expression analysis cDNA synthesis was performed with High Capacity cDNA RT Kit (Life Technologies) and inventoried TaqMan Gene expression assays were used for qPCR together with TaqMan GenEx Master Mix (Life Technologies) following manufacturer’s instructions. The stably expressed human GAPDH gene was identified using the geNorm + module in qbase + and used as reference genes for normalization. Normalized relative expression levels for miRNA and mRNA were calculated using the qbase + software (Biogazelle NV, Zwijnaarde, Belgium).

We employed Exiqon (Woburn, MA) miRCURY LNA™ Universal RT microRNA PCR primer sets because of the exceptional sensitivity and specificity and low background. cDNA was diluted 40-fold with nuclease-free water before PCR amplification. cDNA from human samples were mixed with PCR master mix containing primers for different miR-200 members, UniSp6, or the endogenous reference miRNAs. cDNA from chicken samples were mixed with PCR master mix containing compatible human miR-200a, miR-103a-3p, or chicken (gga)-miR-200b, miR-429, or UniSp6. All PCR reactions were performed in a StepOne Plus or a 7500 real-time PCR instrument with 60 amplification cycles (95 °C for 10 s, followed by 60 °C for 1 min). Melt curve analysis was done to reveal primer dimer formation or non-specific binding. Ct value of each reaction was obtained with threshold set to 5000. Ct values larger than 60 were counted as 60. All reactions were performed in duplicates and the average Ct values were used to compute normalized miRNA level for each human and chicken sample.