Light cycler lc480 real time pcr system

The RNAfast200 kit (Fastagen, Shanghai, China) was applied to HK-2 cell mRNA extraction. SureScript™ First-Strand cDNA Synthesis Kit (GeneCopoeia, United States) was used for reversely transcribing into cDNAs. Real-time PCR experiments were performed using the SsoFast™ EvaGreen Supermix Kit (Bio-Rad, CA, United States) and Light-Cycler LC480 real-time PCR system (Roche, Basel, Switzerland). The RT-PCR reaction temperature was as previously described (Li et al., 2022b (link)). The sequence is as follows: homo GAPDH: Forward 5′-GCA​CCG​TCA​AGG​CTG​AGA​AC -3′, Reverse 5′-TGG​TGA​AGA​CGC​CAG​TGG​A -3’; homo APOC1: forward 5′- CAC​ACT​GGA​GGA​CAA​GGC​TC-3′, Reverse 5′- AAA​CCA​CTC​CCG​CAT​CTT​GG-3’.

The oligonucleotide primers to planking CD44 variable exons and TaqMan probes that are anchor on the border of linked exons were designed (Fig. 1). 10 ng of cDNA used for each amplification of each CD44v with 5 pmoles of each forward and reverse primers and 10 pmoles of FAM-TAMRA labelled oligonucleotide primers (Bioneer, Korea), and 1x TaqMan master mix (Roche, Switzerland) in 10 ul reaction mixture. All the amplifications were carried out triplicate in LightCycler LC480 Realtime PCR system (Roche, Switzerland) with 45 cycles of thermal cycles of 94 °C for 10 sec and 60 °C for 30 sec. Results were analysed by Roche LC480 software v1.5 using absolute quantification by interpolation from the curves of standard from 1.0 × 10² to 1.0 × 10⁹ copies/reaction and non-template control.

Total RNA was isolated using TRI reagent (Sigma Chemicals) and cDNA was generated using Primescript RT master mix (Takara Bio, Inc., Ōtsu, Japan). Duplicate PCR amplifications were carried out using a Light Cycler LC480 Real-Time PCR System (Roche, Basel, Switzerland) with the SYBR Green PCR Kit (Takara Bio, Inc.). mRNA concentrations were calculated using the 2^−ΔΔCt method. The primers used in this study were produced by Sangon Biotech Co., Ltd. (Shangai, China) and the sequences are listed in Table 1.