Hiscript 2 q rt supermix reagent

Quantitative real-time polymerase chain reaction (qRT-PCR) is a useful technique for quantitative analysis of gene expression [38 (link)]. RNA extraction kits (Tian Gen, Beijing, China) were used to extract total RNA. After determination of the RNA concentration with a NanoDrop One Microvolume Spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA), and HiScript II Q RT SuperMix reagent (Vazyme, Nanjing, China) was added to all RNA samples (quantified to 1000 ng) for reverse transcription. The reverse-transcribed samples were cyclically amplified with ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) in a Roche LightCycler 480 (Roche, Basel, Switzerland) with GAPDH as the internal reference gene. The PCR conditions were as follows: 95°C for 30 s followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. Data were processed using the 2^−ΔΔct calculation method. Primers were purchased from Sangon Biotech (Shanghai, China); detailed information regarding the primers is presented in Table 2.

GSE89006 dataset (4 pairs of bladder cancer and NATs) and GSE55433 dataset (tumor = 57, normal = 26) were collected in the GEO database to verify the expression of target lncRNAs.
Next, bladder cancer cells (EJ-1, U3, and 5637) and normal bladder epithelium cells (SV-HUC-1) were purchased from Cell Bank, Institute of Life Sciences, Chinese Academy of Sciences Cell Bank (Shanghai, China). Total RNA was isolated using TRIzol reagent (Bioteke, Beijing, China). Then, reverse transcription was conducted utilizing the HiScript II Q RT SuperMix reagent (Vazyme, Nanjing, China). Subsequently, qRT-PCR was performed by the Hieff TMqPCR SYBR^® Green Master Mix (Yeasen, Shanghai, China) with the primers provided in the Supplementary Table S1. We executed qRT-PCR with the Bio-Rad CFX96 Real-time Quantitative PCR System (Bio-Rad, California, USA).

RNA Keeper-ICE tissue transfer buffer was bought from vazyme company (Nanjing, China). Quick RNA Isolation kit was bought from Biotech Corporation (Beijing, China). AMPure XP beads were bought from Beckman (CA, USA). HiScript® II Q RT SuperMix reagent was bought from vazyme company (Nanjing, China). TRIzol reagent was bought from TransGen Biotech (Beijing, China). Divalent cation fragmentation buffer was bought from Illumina (CA, USA). RNaseH and DNA polymerase I were bought from TaKaRa (Kyoto, Japan). Elution buffer was bought from GENEWIZ (Suzhou, China). Agarose gel electrophoresis buffer was obtained by dissolving 1 g of Agarose in 100 mL of Tris Acetate EDTA electrophoresis buffer. Tris Acetate EDTA electrophoresis buffer was bought from TSINGKE (Beijing, China). Agarose was bought from Sigma (Darmstadt, Germany).