Primescript rt reagent kit gdna eraser

DElncRNAs and immune-related lncRNAs were combined to produce 38 immune-related DElncRNAs, as previously stated. We used TRIzol Reagent (Invitrogen, USA) to extract total RNA from normal and inflamed pulp tissue to validate the identified lncRNAs. The inclusion criteria of samples referred to the published literature [20 (link)]. The detailed information of patients is shown in Table 2. cDNA was synthesized using PrimeScript RT reagent Kit gDNA Eraser (Takara, Japan). qRT-PCR was performed in 20 μL volumes with SYBR Green PCR Master Mix (Roche, USA). The run methods are 95°C for 30 s and 40 cycles at 95°C for 5 s and 60°C for 34 s using QuantStudio Real-Time PCR Systems (Thermo Fishier, USA). By normalizing to the endogenous reference GAPDH, the expression of target genes was estimated. These experiments were approved by the Ethics Committee of West China Hospital of Stomatology, Sichuan University (approval number: WCHSIRB-d-2020-368). The experiments were in complianced with the Minimum Information for the Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines [23 (link)] (Table S1).

Control or PNKP 3′UTR siRNA-transfected and mock or GO/Bleo treated cells were used to assess the depletion of endogenous PNKP by RT-PCR. Total RNA was extracted using TRIzol (ThermoFisher Scientific) according to the manufacturer's protocol. 1 μg of total RNA was used to make cDNA libraries using prime script RT reagent kit gDNA eraser (Takara Bio Inc.) according to the kit's protocol, and RT-PCR was carried out using 1 μl of cDNA to amplify the 3′UTR region of the endogenous PNKP gene, as well as the housekeeping gene (GAPDH) using quick-load Taq 2x master mix (New England Biolabs), according to manufacturer's protocol. The primers used for amplifying the 3′UTR region of PNKP and GAPDH are listed in Table 2. The amplified PCR products were run in 1.5% agarose gel and stained with ethidium bromide, and the image was processed by Gel Doc EZ imager (BioRad). The quantitation of gel bands was performed using ImageJ software and the endogenous PNKP expression was normalized using GAPDH. The relative expression levels were presented with the expression of endogenous PNKP in control siRNA transfected cells considered as 100.

Total RNA from formalin-fixed paraffin-embedded tissues was extracted by using nucleic acid extraction kit (AmoyDx, China) according to the manufacturer’s protocol. And total RNA was extracted from cultured cells by using the RNAiso plus reagent (TaKaRa, Japan). Total RNA was reverse transcribed by using Primescript™ RT reagent kit gDNA Eraser (TaKaRa, Japan). qPCR was performed by using SYBR Premis Ex Tag™II(TaKaRa, Japan) on the 7500 qPCR System (Applied Biosystems Inc., Foster City, CA, USA).The qRCR reactions were pre-incubated for 10 min at 95 °C, followed by 40 cycles of denaturation for 30 s at 95 °C and annealing for 1 min at 60 °C. The primers for amplifying GOLPH3 are as follows: 5’-GGGCGACTCCAAGGAAAC-3′ (forward) and 5’-CAGCCACGTAATCCAGATGAT-3′ (reverse), and primers to amplify GAPDH contains: 5’-AGCCACATCGCTCAGACACC-3′ (forward) and 5’-CGCCCAATACGACCAAATCC-3′ (reverse). Reactions containing either no reverse transcriptase or no template were used as negative controls, and all reactions were performed in triplicate. The GAPDH was used as the normalization control, and the relative expression level was calculated using the 2 − ^ΔΔCT equation.