Primescript 2 1st strand synthesis kit

In the clinical validation set, we chose CRC patients based on the following criteria: (1) patients treated in the Affiliated Hospital of Jiangnan University; (2) patients with complete follow-up information, including survival status and survival time; (3) patients who did not receive treatment before surgery. Eighty-five CRC patients were gained from the Affiliated Hospital of Jiangnan University as the clinical validation set. The clinical information of all patients was listed in Table 1. All patients signed the informed consent about using their tumor tissues, and this study was ratified by the clinical research ethics committees of the participating institutions.
Total RNA of tumor tissues was isolated using RNAiso Plus (Takara, Japan). NanoDrop 2000 (Thermo, USA) was used to measure RNA concentrations. Total RNA was reverse transcribed to complementary DNA (cDNA) using the Prime-Script II 1st Strand Synthesis Kit (TaKaRa). The expression levels of lncRNAs were quantitated using the UltraSYBR Mixture (CWBIO, China) by the ViiA7 real-time PCR system. The lncRNA expression level was calculated as follows: ΔCT = CT (lncRNA) – CT (β-actin). The sequences of used primers are listed in Supplementary Table 1.

We applied the PrimeScript II 1st Strand Synthesis Kit (TaKaRa Biotechnology, Dalian, China) to transcribe the total RNA reversely into cDNA, and conducted qRT-PCR on the ViiA7 real-time PCR system (Applied Biosystems, Grand Island, NY, USA) with the RT-RNA PCR kit (TaKaRa). Furthermore, we normalized the relative gene expression to β-actin and used the 2^-∆∆Ct method to do the calculation.

Eyes were homogenized using MixerMill MM300 (Reche, Haan, Germany), and total RNA was isolated using ISOGEN2 reagent (Wako). Total RNA was treated with RNase‐free DNase (TaKaRa, Otsu, Japan) to eliminate genomic DNA contamination and then purified by phenol–chloroform extraction, followed by isopropanol precipitation. First‐strand cDNA was prepared using the PrimeScript II 1st Strand Synthesis kit (TaKaRa). Genomic DNA was isolated from the muscle tissue according to a previously described method (Sambrook & Russell, 2001). Polymerase chain reaction (PCR) to amplify cDNA and genomic DNA was performed using the TaKaRa Ex Taq Hot Start Version kit (TaKaRa) under conventional conditions. Amplified DNA fragments were purified using the Nucleospin gel and PCR cleanup kit (Macherey‐Nagel, Düren, Germany) after agarose gel electrophoresis. Custom oligonucleotides designed for PCR (Table S1) were synthesized at Life Technologies (Carlsbad, CA, USA). The full‐length cDNA fragments were subcloned into pGEM‐T Easy vectors (Promega, Fitchburg, WI, USA) followed by standard alkaline‐SDS plasmid preparation (Sambrook & Russell, 2001).

Total RNA was extracted from cultured cells using TRIzol reagent (R401–01, Vazyme) and reverse transcribed into complementary DNA using the PrimeScript II 1st Strand Synthesis Kit (6210A, TaKaRa, Japan). qRT-PCR was performed on the ViiA7 real-time PCR system using the UltraSYBR Mixture (CW0957M, CWBIO) . The real-time PCR primers used are as follows: SIRT3 (forward): 5ʹ-CAGTCTGCCAAAGACCCTTC-3ʹ; SIRT3 (reverse): 5ʹ-AACACAA TGTCGGGCTTCAC-3ʹ; Actin (forward): 5ʹ-TCCATCATGAAGTGTGACG-3ʹ; Actin (reverse): 5ʹ-TACTCCTGCTTGCTGATCCAC-3ʹ.

Whole RNA was extracted from cancer cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of the extracted RNA into cDNA was achieved using the PrimeScript™ II 1st Strand Synthesis Kit (Takara, Kusatsu, Japan). The acquired complementary DNA replicates were further analyzed using SYBR Green Master Mix (Takara, Kusatsu, Japan) following the reaction conditions of 95 °C (300 s), followed by 45 cycles of 95 °C for 5 s, 58 °C for 30 s, 95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s. Quantitative real-time PCR was performed using the QuantStudio3 PCR system (ThermoFisher, Massachusetts, USA), and relative mRNA abundance was calculated using the 2^−ddCt method.