Sybr premix ex taqtm 2 reagent kit

Total RNA was extracted from the obtained tissues and cultured cells using TRIzol reagent (Invitrogen). The purity and concentration of the extracted RNA was evaluated using a Nanodrop 3000 (ThermoFisher, Scotts Valley, CA, USA). A high-capacity cDNA reverse transcription kit (Takara, Kyoto, Japan) was used to synthesize complementary DNA. Then, qRT-PCR was performed using SYBR® Premix Ex TaqTM II reagent kit (Takara) on a ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for 45 cycles (95 °C for 3 min, 95 °C for 15 s, and 60 °C for 1 min). U6 was used as internal control for miR-452-3p and β-actin for all others. The primers used are listed in Supplementary Table 1, which were synthesized by Shanghai Sangon Biotech Co., Ltd (Shanghai, China). The specificity of all PCR products was assessed by melting curve analysis. Relative gene expression was analyzed using the 2^−ΔΔCt method.

A MiRNeasy Mini Kits (217004, QIAGEN, Hilden, Germany) were utilized to extract the total RNA content from the cells after transfection. The obtained total RNA was subsequently reversely transcribed into complementary DNA (cDNA) (10 μL) with a PrimeScript RT kit (RR036A, Takara Biotechnology Ltd., Dalian, Liaoning Province, China). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using a SYBR® Premix Ex TaqTM II reagent kit (RR820A, Takara) on an ABI7500 real-time PCR instrument (Applied Biosystems, Carlsbad, CA, USA). The primer sequences of DPP10-AS1, miR-127-3p, ADCY1, CD133, CD44, Lgr5, ADLH1, U6 (as the internal control for miR-127-3p) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as the internal control for DPP10-AS1 and other genes) were synthesized by Takara (Table 2). The 2^-ΔΔCt method was performed to determine the relative expression levels. Each experiment was repeated three times.

Total RNA was obtained from cultured cells and the tissues using TRIzol reagent according to the manufacturer’s instructions (ThermoFisher). The purity of the extracted RNA was assessed using a Nanodrop 3000 (ThermoFisher). The first strand of complementary DNA was synthesized using a high-capacity cDNA reverse transcription kit (Takara, Kyoto, Japan). Then, qRT-PCR was conducted utilizing SYBR^® Premix Ex TaqTM II reagent kit (Takara) on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for 40 cycles (95 °C for 3 min, 90 °C for 15 s, and 60 °C for 1 min). The primer sequences for qRT-PCR are listed in Supplementary Table 1, which were designed and synthesized by Sangon (Shanghai, China). U6 was selected as internal control for miR-155-5p and β-actin for all others. The specificity of all PCR products was checked using melting curve analysis. Relative gene expression was determined by the 2^−ΔΔCt method.

The TRIzol reagent kit (Invitrogen, CA, USA) was used to extract total RNA from the obtained tissues and cultured cells. The extracted RNA was then purified and quantified using the Nanodrop 3000 (ThermoFisher, Scotts Valley, CA, USA). The first-strand complementary DNA was synthesized utilizing a high-capacity cDNA reverse transcription kit (Takara, Kyoto, Japan). qRT-PCR was conducted using SYBR® Premix Ex TaqTM II reagent kit (Takara) on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for 40 cycles (95°C for 2 min, 95°C for 10 sec, and 60°C for 30 sec). Melting curve analysis was used to evaluate the specificity of all PCR products. Relative gene expression was quantitated using the 2^−ΔΔCt method. The sequences of mRNA primers were designed and synthesized by Shanghai Sangon Biotech Co., Ltd (Shanghai, China). The primers were listed in Supplementary Table 1. β-Actin was used as an internal control.

After being anesthetized using pentobarbital sodium, brain tissues were collected. Next, the brain tissues of the mice were homogenized by TRIzol reagent (Invitrogen, USA) on ice to extract total RNA. Afterwards, the extracted RNA was reverse transcribed into cDNA utilizing High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). RT-qPCR analysis was conducted employing a SYBR^® Premix Ex TaqTM II reagent kit (RR820A, Takara) on a 7500 Real-Time PCR System (Applied Biosystems). U6 served as the endogenous control for miR-485-5p. The detection results for mRNAs (PACS1, MON2, TCTA, SOX10, and PPARGC1A) were normalized to GAPDH. Expression fold changes were calculated adopting the 2^−ΔΔCt method. The raw data of the 2^−ΔΔCt can be found in Table S1.