Quantmir rt kit

Total RNA from cell lines and tissues was extracted with TRIzol^® reagent (Invitrogen), according to the manufacturer’s instructions. The concentration of the total RNA was quantified using a NanoDrop-2000 (Peqlab). cDNA was synthesized with the TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and expression was determined using the QuantMir RT Kit (System Biosciences, Mountain View, CA, USA) and ABI 7900 Sequence Detection System (Applied Biosystems) using miR-506 and U6 primers (Applied Biosystems). To evaluate ZEB2 mRNA expression, RNA was reverse-transcribed into cDNA by the Primescript^™ RT reagent kit (Takara, Dalian, China) and examined with the Real-time PCR Mixture Reagent (Takara) and ABI 7900 Sequence Detection System (Applied Biosystems), using primers for ZEB2 and α-tubulin. The relative miR-506 expression was normalized to U6 expression and the expression of ZEB2 mRNA was normalized to α-tubulin using the 2-ΔΔCt method. Values of genes were first normalized against U6 or α-tubulin, and then compared to the experimental controls.

Total RNA sample was used to reversely transcribe miRNAs to a strand cDNA using a TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA), and then was quantified by using the QuantMir RT Kit (System Biosciences, Mountain View, CA) in ABI 7900 Sequence Detection System (Applied Biosystems) with miR-449a und U6 primers (Applied Biosystems). The expression levels of each miRNAs were normalized against U6 expression. The relative expression levels of serum miR-449a was quantitatively analyzed by using the 2–^ΔΔCT method.