Sybr qpcr kit

Total RNA was extracted from tissue with TRNzol Reagent (TIANGEN, Catalog: DP405), according to the manufacturer’s instruction. Complementary DNA (cDNA) was synthesized with PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara, Catalog: RR047A). Gene expressions were measured by the StepOne Plus Real-Time PCR system (Applied Biosystems) with SYBR qPCR kit (Takara, Catalog: RR820L). Gapdh was used as an internal control.

Total RNA from each sample was extracted using Trizol reagent. RNA quality and purity were determined using 2% agarose gel electrophoresis and ultraviolet spectrophotometry, respectively. The reverse-transcribed cDNA was synthesized using the Prime Script RT Reagent Kit and stored at -20°C. Primers were designed using the Primer 3.0 online program based on the CDS sequence of NtCXE genes for quantitative real-time (qRT)-PCR. An Applied Biosystems CFX96 machine was used for the qRT-PCR with the SYBR qPCR kit (TaKaRa). The tobacco ribosomal protein gene, L25 (GenBank No. L18908), was used as an internal reference, and three biological replicates were performed (Schmidt and Delaney, 2010 (link)). The gene primers used in this study are listed in Supplementary Table 2.

The expression of the selected microRNA was determined by SYBR qPCR Kit (Takara, Dalian, China) according to the manufacture’s protocol with 2.0 μL cDNA as template. The primer sequences of miRNAs used in Real-Time PCR were listed in Supporting Information (Additional file 1: Table S1 and Figure S1-S3). The reaction was performed with the following program: 95 °C for 10 s, 40 cycles of 95 °C for 5 s, 60 °C for 20 s, and followed by the thermal denaturing step to generate the dissociation curves to verify amplification specificity. Cel-miR-39 was served as the normalization control, and data were analyzed by Bio-Rad CFX Manager software (Bio-Rad, CA, USA) to obtain miRNAs relative expression scores. Cycle threshold (Ct) values of each miRNA were normalized to cel-miR-39-3p, and the 2^−∆∆Ct method was used to analyzed the relative expression level of miRNA.

The two human hepatoblastoma cell lines and normal control cell line were used to verify the expression of the identified immune-related lncRNAs. Chronic HBV-producing HB611 cells were purchased from Keibai Academy of Science (Nanjing, China) and cultured in DMEM medium (Corning, NY, USA) plus 10% fetal bovine serum (FBS, Gibco). The hepatocellular carcinoma Hep3B cell line, which contains an integrated hepatitis B virus (HBV) genome and constitutively secretes HBsAg, was also purchased from Keibai Academy of Science (Nanjing, China) and cultured in RPMI-1640 medium (Corning, NY, USA) plus 10% fetal bovine serum (FBS, Gibco). The normal hepatocytes HL7702 were purchased from ScienCell Research Laboratory (Shanghai, China) and cultured in RPMI-1640 medium (Corning, NY, USA) plus 10% fetal bovine serum (FBS, Gibco). The total RNA of those human hepatoblastoma cell lines were extracted with TRIzol Reagent (Invitrogen) and do qRT-PCR analysis with SYBR qPCR kit (TaKaRa). All the primers (forward and reverse primers) were synthesized by Sangon Biotech (Shanghai, China).