Qrt pcr

Total RNA was extracted from the samples using RNA extraction reagent RNAiso (TaKaRa, Japan) on the basis of the protocol of manufacture. Total RNA was dissolved in the RNase free water (Sangon, China). The concentration was determined by NanoDrop 2000 spectrophotometer (Thermo company, USA), and the purity was detected through 1% agarose gel electrophoresis. First-strand cDNA used for gene cloning was synthesized using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan), and the PrimeScript™ RT Master Mix was used to synthesize cDNA template for quantitative Real Time PCR (qRT-PCR) (Vazyme, China). The reverse transcription products were stored at -20°C until used.

Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, USA), while cytoplasmic and nuclear RNA were extracted using cytoplasmic & nuclear RNA purification (Amyjet Scientific, Wuhan, China). qRT-PCR was performed following the protocol (Vazyme, Nanjing, China).

Subcellular fractionation of HPC cells was performed using Cytoplasmic & Nuclear RNA Purification Kit (21000, Norgen Biotek, Canada). Total RNAs were subsequently extracted from both nuclear and cytoplasmic fractions. Then equal volumes of RNA from both fractions were subjected to qRT-PCR (Q311-02, Vazyme, Nanjing) analysis.