Mipure cell tissue mirna kit

Total RNA was extracted using MiPure Cell/Tissue miRNA Kit (Vazyme, Nanjing, China, 21000) and reverse transcribed (Invitrogen, Carlsbad, CA, USA) at 48 h after cell transfection. Quantitative real-time PCR (qRT-PCR) was performed and GAPDH was used to normalize the expression levels of lncRNA and. The primers were listed as Table S4.

Total microRNAs were extracted using MiPure Cell/Tissue miRNA Kit (RC201, Vazyme). C19MC microRNAs (miR-517c-3p, miR-517-5p, miR-525-3p) and U6 were reverse transcribed into complementary DNA using miRNA 1st Strand cDNA Synthesis Kit (MR101, Vazyme). Then, 2 µl of cDNA corresponding to C19MC microRNAs or U6 were mixed with components of miRNA Universal SYBR qPCR Master Mix. The analysis was performed using a 7500 Real-Time PCR system under the conditions described according to the specification. The expression of C19MC microRNAs and U6 were determined using the comparative CT method relative to the expression in the reference sample. All the primers used were also described in supplementary Table 1.

The same protocols provided in 2.4 were performed for quantitative real-time PCR (RT-qPCR) validation. MiPure Cell/Tissue miRNA Kit (Vazyme) was used to collect and extract samples according to the manufacturer's instructions. HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) was used for validation of RNA biomarker expressions. Primers shown in Table S4 were designed to validate candidate genes using Primer Premier 5.1. A relative quantitative method was applied to calculate the fold changes (log₂FC=-ΔΔCT) of mRNA expression relative to the reference genes (16S rRNA).

Total RNA was isolated from cells and tissues by using TRIzol reagent (Invitrogen). The genomic DNA was eliminated with TURBO DNA-free Kit (Invitrogen, AM1907) as per the manufacturer’s instructions. RNA quality was assessed by using NanoDrop 2,000 (Thermo Scientific). The cDNAs were synthesized by ReverTra Ace qPCR RT Master Mix (Toyobo, FSQ-201) or First-Strand cDNA Synthesis Kit (Vazyme, R211-01). qPCR was performed using SYBR Green Supermix (Bio-Rad, 172-5124). Primers for qPCR are listed in Table 1The method for stem-loop real-time quantification of miRNA was described previously (33 (link)), and the schematic diagram is shown in Fig. S10. For miRNA quantification, total RNA from cells was purified using the MiPure Cell/Tissue miRNA Kit (Vazyme, RC201) to retain small RNA according to the manufacturer’s protocol. The cDNAs were synthesized by miRNA 1st Strand cDNA Synthesis Kit (Vazyme, MR101-01). Stem-loop qPCR was performed using ChamQ Geno-SNP Probe Master Mix (Vazyme, Q811). The primers and probes for stem-loop qPCR are listed in Table 2

Total RNA was extracted from cells by FastPure Cell/Tissue Total RNA Isolation Kit and MiPure Cell/Tissue miRNA Kit (Vazyme, Nanjing, China), and then it was reverse-transcribed to cDNA using the HiScript II Q RT SuperMix for qPCR Kit and miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, Nanjing, China). The cDNA was used as a template for real-time qPCR; and qRT-PCR was performed on a QuantStudio3 Real-Time PCR System (Applied Biosystems, USA) with ChamQ™ Universal SYBR^® qPCR Master Mix (Vazyme, Nanjing, China). β-Actin and U6 were used as loading controls for GOLPH3, TUG1, and miR-142-5p. The relative fold change in expression of the target normalized to expression of the corresponding control was calculated by the comparative Ct method. The primers used in this study are shown as follows: GOLPH3 (forward: 5′-GGGCGACTCCAAGGAAAC-3′ and reverse: 5′-CAGCCACGTAATCCAGATGAT-3′), miR-142-5p (forward: 5′-GCGCGCATAAAGTAGAAAGC-3′ and reverse: 5′-AGTGCAGGGTCCGAGGTATT-3′), TUG1 (forward: 5′-TAGCAGTTCCCCAATCCTTG-3′ and reverse: 5′-CACAAAT TCCCATCATTCC-3′), β-actin (forward: 5′-GCCGACAGG ATGCAGAAGG-3′ and reverse: 5′-TGGAAGGTGGACAGCG AGG-3′), U6 (forward: 5′-GCTTCGGCAGCACATATACTAA AAT-3′ and reverse: 5′-CGCTTCACGAATTTGCGTGTCAT-3′), and GOLPH3-3′UTR (forward: 5′-GCGCCCGCGATCGCG AATTCCTCTGCTCGGGGTGAAC-3′ and reverse: 5′-ACTAGTCTCGAGTTCAGGCACTTTCAAGATCATTG-3′).