Paris kit

Total RNA extraction from GC cells or clinical samples was performed using the TRIzol reagent (Invitrogen, USA) as previously described [12 (link)]. For SNHG22 distribution analysis, PARISTM Kit (Thermo Fisher, USA) was used to extract the nuclear and cytoplasmic RNA separately as previously described. Quantitative RT-PCR was carried out using SYBR Green PCR Master Mix (Vazyme) with an ABI Prism 7900 Sequence detection system (Applied Biosystems, Canada). U6 and beta-actin were used as endogenous controls. The primers are shown in Supplementary Table 1.

The content of RNA in the cytoplasm and nucleus was analyzed with PARISTM Kit (Thermo Fisher AM1921). When the confluence rate was >90% in the 60 mm dish, the cells were collected. Cytoplasm and nuclear materials were separated by centrifugation following the manual of kit. The cytoplasm supernatant and nucleus precipitate were transferred to different tubes with equal volumes of 2× Lysis Binding Buffer and mixed thoroughly with a pipette. The obtained RNA was stored at -80°C with an appropriate amount of DNAase to prevent degradation.

Total tissue and cellular RNA were extracted with TRIzol Reagent (Vazyme, Nanjing, China). PARIS^TM Kit (ThermoFisher Scientific, Waltham, MA, USA) was utilized to obtain cytoplasmic and nuclear RNA fractions in line with specific protocols. Total RNA (approximately 10 μg) was subject to incubation using RNase R (40 U, Epicentre Technologies, Madison, USA). HiScript QRT SuperMix from qPCR Kit (Vazyme, Nanjing, China) was thereafter adopted for reverse transcription of RNA. TransStart Top Green qPCR Super Mix (TRAN, China) was utilized for qRT-PCR amplification using the ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The 2^−ΔΔCT method was later adopted for data quantification, with GAPDH being the endogenous reference.

RNA from separate nuclear and cytoplasmic fractions was isolated using PARIS^TM Kit (Thermo Fisher Scientific). Total RNA was extracted and subjected to reverse transcription using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan). For mRNA and circRNA, a mixture of random and oligo d(T) primers were employed in reverse transcription. For miR-1301-3p, random primers and 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGG ATACGACGAAGTC-3′were used in the reaction system. gDNA was isolated using a TIANamp Genomic DNA Kit (Tiangen).
The Premix Taq DNA Polymerase (TaKaRa) was used for PCR. The cDNA and gDNA PCR products were analyzed using 2.5% agarose gel electrophoresis. Quantitative real-time PCR was performed using the SYBR Premix Ex Taq II (TaKaRa). Relative mRNA and circRNA levels were normalized to GAPDH. In the control group, the gene expression was calibrated as 1. For miRNAs, U6 was used as the internal control. The primers used in PCR and qPCR analyses are summarized in Supplementary Table S2. All the primers span multiple introns and/or exons, and were validated by melting curve analysis.

Cytoplasmic and nuclear RNA were extracted from PPs using PARIS^TM kit (AM1921, Thermo Fisher Scientific) according to the manufacturer's instructions. RNA extracted from the two fractions was evaluated by qRT‐PCR. Data were analyzed to evaluate the percentages of nuclear and cytoplasmic RNA.