Power sybr green

RNA was reverse transcribed to cDNA using a specific Reverse Transcription Kit for miRNA (D350, Takara, Tokyo, Japan), and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted with Power SYBR Green (DRR081A, Takara, Tokyo, Japan). The forward primers for the miRNA amplification were designed based on the mature miRNA sequences (Table 1), and the reverse primers were provided by the manufacturer of the kit. MiRNA levels were quantified using ViiA7 (Applied Biosystems, CA, USA) with the 2^-ΔΔct relative quantification method [19 (link)].

Total RNA was extracted from frozen tissues or cultured cells using TRIZOL reagent (Invitrogen, Carlsbad, CA). For qRT-PCR, the isolated RNA was reverse transcribed to cDNA using a Reverse Transcription Kit (Takara, Dalian, China). Real-time PCR analyses were conducted with Power SYBR Green (Takara, Dalian, China). The results were normalized to the expression of GAPDH. The primers were as follows: GAPDH sense 5′-GGGAGCCAAAAGGGTCAT-3′, reverse 5′-GAGTCCTTCCACGATACCAA-3′; HNF1-AS1 sense, 5′-TCAAGAAATGGTGGCTAT-3′, reverse 5′-GCTCTGAGACTGGCTGAA-3′. qRT-PCR and data collection were performed using an ABI 7500 instrument (Applied Biosystems, Foster City, CA). All qRT-PCR reactions were performed in duplicate.

Isolated RNA was reverse-transcribed to cDNA using a Reverse Transcription Kit (Takara, Dalian, China). The qRT-PCR analyses were performed using a StepOnePlus RT-PCR Instrument with Power SYBR Green (Takara, Dalian China). The qRT-PCR conditions were as follows: 95 °C for 2 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds. All experiments were performed and analyzed in triplicate. The primers used in this study were listed in Table S1. Then, lncRNA expression levels were normalized to GAPDH and calculated using the 2^−ΔΔCt method.

Extracted RNA (100 ng) was reverse-transcribed to first-strand cDNA using the High Capacity cDNA Reverse Transcription Kit (Takara, Dalian, China). Then, qPCR was performed using Power SYBR Green (TaKaRa, Dalian, China) on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, USA), which was also used for data collection. The thermal cycling program used for quantification was as follows: an initial denaturation step at 95°C for 15 sec, followed by 40 cycles of 95°C for 5 sec and 58°C for 34 sec, with melting curve analysis. Each measurement was performed in triplicate to remove any outliers. Each RT-qPCR run included a calibrator (expression level of CRNDE-h in HT-29 cells) and a negative control lacking cDNA. Relative expression was calculated according to comparative Quantification cycle (Cq) method. The levels of tissue lncRNA CRNDE-h were normalized using GAPDH, as recommended in other studies. And, exosomal CRNDE-h expression was recorded as the ratio against the geometric mean of GAPDH and UBC [29 (link)]. Primer sequence was shown in Supplementary Table S1According to the formula E=10^-1/slope-1, we used the slope of the standard curve of cDNA to calculate the reaction efficiency (E).

Total RNA was extracted from the LA and the LSG using TRIzol reagent, according to the manufacturer’s protocol. Then, this was treated with DNase I to degrade any trace of DNA, and purified using an RNeasy Kit (Takara, Tokyo, Japan). The integrity of each sample was confirmed by agarose gel analysis. Total RNA was reverse transcribed using TaqMan reverse transcription reagents (Takara). The expression levels of candidate genes were measured by real-time quantitative RT-PCR (qRT-PCR) using Power SYBR Green (Takara) dye, and quantified using ViiA7 (Applied Biosystems, CA, USA). In each assay, both an endogenous control gene (β-actin) and a target gene from the same samples were amplified in duplicate in separate tubes. The mRNA levels of each target gene were calculated using the relative standard curve method and normalized against the corresponding β-actin mRNA levels. The expected amplicon sizes were confirmed by gel electrophoresis. The sequences of the genes studied were obtained from GenBank, and the primers were designed using Primer Premier 5.0 software (Premier Biosoft). Table 1 presents the primer sequences and amplicon sizes of the selected genes.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to determine the mRNA expression levels of genes. Briefly, total RNA was extracted from cells by using the TRIzol reagent (Invitrogen, China) according to the manufacturer's instructions. RNA was reverse transcribed to cDNA by using a Reverse Transcription Kit (Takara, China). Real-time PCR analyses were performed with Power SYBR Green (Takara, China), and the primers were synthesized at Invitrogen, China. The primers for each gene were based on a previous report [24 (link)]. Results were normalized to the expression of the human β-actin gene.
For the Western Blot analysis, the total protein was lysed in a radioimmunoprecipitation assay buffer (RIPA buffer, Solarbio, China). Samples were prepared under reducing conditions by using SDS-PAGE gels before being blotted and detected using an anti-KDM4C antibody (Abcam, China), OCT-4 (Abcam, China), and α-tubulin (Abcam, China).

Total RNA from samples was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., USA). The PrimeScript RT Reagent kit (Takara Biotechnology) was employed for cDNA synthesis through reverse transcription. Subsequently, Power SYBR Green (Takara Biotechnology) and the RT-PCR detection system were utilized to amplify specific cDNA fragments with corresponding primers. Data were expressed as relative levels. The 2-ΔΔCq method was used to determine relative gene expression, using β-actin as an internal reference. Furthermore, the primer sequences employed in the RT-PCR experiments are provided in Additional file 1: Table S4.