Primescript qrt pcr kit

This procedure was performed according to a protocol in a previously published report^{26 (link)}. BMDMs were washed twice with PBS and lysed in lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM dithiothreitol (DTT), 1:100 protease inhibitor cocktail and 400 U/ml RNase inhibitor). The cell lysates were centrifuged. A 50-μl aliquot of cell lysate was saved as the input, and the remaining sample was incubated with 20 µl of protein A beads previously bound to an IgG antibody or anti-YTHDF1 antibody (Proteintech) for 4 h at 4 °C. The beads were washed two times with wash buffer (50 mM Tris, 200 mM NaCl, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, and an RNase inhibitor). RNA was eluted from the beads with 50 μl of RLT buffer and purified with Qiagen RNeasy columns. RNA was eluted in 100 µl of RNase-free water and reverse transcribed into cDNA using a PrimeScript qRT-PCR kit (Takara) according to the manufacturer’s instructions. The fold enrichment was detected by qRT-PCR. The primers used for testing Spred2 mRNA levels are listed in Supplementary Table 3.

This procedure was performed according to a previously published report^{24 (link)}. Hepa 1-6 cells or mettl3-HET/cKO hepatocytes were washed twice with PBS and lysed in lysis buffer (150 mM KCl, 10 mM HEPES (pH 7.6), 2 mM EDTA, 0.5% NP-40, 0.5 mM dithiothreitol (DTT), 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor). The cell lysates were centrifuged. A 50-μl aliquot of the cell lysate was saved as input, and the remaining sample was incubated with 20 µl of protein A beads previously bound to 5 μg IgG or an anti-insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) antibody (Proteintech^TM) for 4 h at 4 °C. The beads were washed 2 times with wash buffer (50 mM Tris, 200 mM NaCl, 2 mM EDTA, 0.05% NP40, 0.5 mM DTT, RNase inhibitor). RNA was eluted from the beads with 50 μl of RLT buffer and purified with RNeasy columns (217004, QIAGEN). RNA was eluted in 100 µl of RNase-free water and reverse transcribed into cDNA using a Prime Script qRT-PCR Kit (Takara) according to the manufacturer’s instructions. The fold enrichment was determined by qRT-PCR. The primers used for amplifying mouse mature Gys2 and Fasn mRNA are listed in Supplementary Table 2.

Total RNA was extracted from WT and Tg mouse colonic tissues or HT-29 cells following the manufacturer's guidelines and reversed transcribed into cDNA by PrimeScript qRT-PCR Kit (TaKaRa, China). The RT-PCR analysis was performed using SYBR® GreenER™ qPCR SuperMix kit (Invitrogen, Carlsbad, CA) on a 7500 Real-time system (Applied Biosystems, USA). Primer sequences are listed in Supplementary Table 2. GAPDH was used as an internal standard. Data were calculated by the comparative cycle threshold (CT) (2^−ΔΔCT) method.

Total RNA was isolated from cells using TRIzol (Invitrogen). For qRT-PCR analysis of nascent and mature mRNAs, first-strand cDNA was synthesized using a Prime Script qRT-PCR Kit (Takara). The amplification signal of qPCR data was acquired by Bio-Rad CFX Manager 3.1. The expression levels of the target genes were determined by amplification with specific primers. The primers used are listed in Supplementary Table 2.

For determination of mRNA stability, BMDMs were treated with actinomycin D (Sigma) at a final concentration of 5 μg/ml for 0, 3, or 6 h. The cells were then collected, and total RNA was extracted for reverse transcription using a PrimeScript qRT-PCR kit (TaKaRa). The mRNA transcript levels of interest were determined by qRT-PCR.

Total RNA was isolated from HCC tissues and cell lines using TRIzol RNA isolation reagent (Takara, Japan) and reverse transcribed using a PrimeScript qRT-PCR kit (Takara, Japan) according to the manufacturer’s instructions. QRT-PCR was performed with an SYBR(R) PrimeScript RT-PCR Kit (Takara, Japan) using an ABI7500 system (Applied Biosystems, USA) and with specific primers: LOXL4 [forward 5’-CCGCTGCAAGTATGATGG-3′; reverse 5’-GTTCCTGAGACGCTGTTC-3′], 18sRNA [forward 5’-TGCGAGTACTCAACACCAACA-3′; reverse 5’-GCATATCTTCGGCCCACA-3′]. Relative LOXL4 gene expression analysis was performed using the eq. 2^-ΔCt [ΔCt = Ct (LOXL4) – Ct (18sRNA)], with 18sRNA used as an internal control.

This procedure was performed according to a previously published report [36 (link)]. BMDMs were washed twice with PBS and lysed in lysis buffer (150 mM KCl, 10 mM HEPES (pH 7.6), 0.5% NP-40, 2 mM EDTA, 0.5 mM dithiothreitol (DTT), protease inhibitor cocktail, 400 U/ml RNase inhibitor). The cell lysates were centrifuged to obtain the supernatant. A 50-μl aliquot of cell lysate was saved to serve as input, and the remaining lysate was incubated with 20 μl of protein A beads, previously bound to IgG antibody or anti-YTHDF1 antibody (Proteintech) for 4 h at 4°C. The beads were then washed 4 times with washing buffer (50 mM Tris, 200 mM NaCl, 2 mM EDTA, 0.05% NP40, 0.5 mM DTT, RNase inhibitor). RNA was eluted from the beads with 50 μl of elution buffer (5 mM Tris-HCL (pH 7.5), 1 mM EDTA, 0.05% SDS, 20 mg/ml Proteinase K) for 2 h at 50°C, and purified with Qiagen RNeasy columns. RNA was eluted in 100 μl of RNase-free water and were reverse transcribed into cDNA using a PrimeScript qRT-PCR kit (TaKaRa) according to manufacturer’s instructions. The fold enrichment was measured by qRT-PCR. The primers used for testing Dnmt3a mRNA are listed in S2 Table.