Taqman human mirnas qpcr quantitation kit

Total RNA was extracted from cell lines and paraffinized tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and the High Pure FFPE RNA Micro Kit (Roche, Basle Switzerland) according to the manufacturer's instructions. After synthesizing cDNA, miR-520g was amplified using real-time PCR, with the TaqMan Human miRNAs qPCR Quantitation Kit (Applied Biosystems, Foster City, CA, USA) used for quantification following amplification. The small nuclear RNA U6 was used as control. The Quantitative SYBR Green PCR Kit (Qiagen, Hilden, Germany) was used to quantify DAPK2 mRNA using GAPDH as the internal control. The primers used in this study were as reported previously [16 (link)]. All experiments were performed in at least triplicate.

The tissue specimens obtained from CRC patients after surgery were fixed in 10% formalin and embedded in paraffin. Total RNA were extracted from CRC cells using Trizol reagent (Invitrogen) and paraffin-embedded tissues using High Pure FFPE RNA Micro Kit (Roche) according to the manufacturer's instructions, respectively. After total RNA were reverse transcripted into complementary DNA which acted as the template for the amplification of RNA, real-time PCR was used for miR-20a-5p amplification and TaqMan Human miRNAs qPCR Quantitation Kit (Applied Biosystems, Foster City, CA, USA) for quantification following amplification. The small nuclear RNA U6 was used as normalized control. The Quantitative SYBR Green PCR Kit (Qiagen, Hilden, Germany) was used to quantify the Smad4 mRNA, E-cadherin and N-cadherin expression level respectively, GAPDH was used as the internal control. All the experiments were repeated at least triplicates [34 (link), 37 (link)].