Bugle loop mirna qpcr kit

Total RNA was extracted using Trizol reagent (Takara), cDNA was reverse transcribed using PrimeScript™ RT Reagent Kit (Takara), and miR‐148b‐3p and U6 were reverse transcribed using Bugle‐Loop™ miRNA qPCR kit (RiboBio). Expression of RNA was detected using SYBR® PrimeScript™ RT‐PCR Kit (Takara, Japan). β‐actin was used as the internal reference for lncRNA and mRNA, and U6 was used as the internal reference for miRNA. Expression levels were quantitated using the 2^‐ΔΔCt method. RiboBio designed and synthesized all miRNA and U6 primers. ABM (Canada) synthesized all other primers used in this study. The primer sequences are listed in Table S1.

Trizol reagent (Invitrogen) was applied to extract total RNA from tissue samples and HCC cells. The transcript abundance of circKCNN2 and genes were quantified by using PrimeScript RT Master Mix (Takara, Dalian, China) and TB Green Premix Ex Taq II (Takara), respectively. The Bugle‐Loop™ miRNA qPCR kit (RiboBio) was applied to quantify miRNA‐520c‐3p. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) served as internal reference for the quantification of gene and circRNA while U6 was applied as the internal reference for the quantification of miRNA. All the primers used for RT‐qPCR assays are listed in Table S6. Western blot and IHC assays were conducted as previously described.²⁰ The primary antibodies used in this study were as follows: MBD2 (1:1000, # A301633A, Bethyl, Montgomery, TX), FGFR4 (1:1000, #8562, CST, Danvers, MA), FGF19 (1:1000, #83348, CST), FRS2 (1:1000, #MAB4069, R&D Systems, Minneapolis, MN), and GAPDH (1:1000, #3683s, CST). Western blot analyses were repeated three times. GAPDH was used as the loading control and normalization control. ImageJ software (version 1.51, https://imagej.nih.gov/ij/) was applied to quantify the signal strength of the bands.