Twelve DEGs (relative fold changes were above 4Log2) were chosen randomly to verify the data of RNA-seq by RT-qPCR. Primers sequences used for RT-qPCR (http://www.ncbi.nlm.nih.gov/ ) were synthesized by Sangon Biotech (seen in Table 1 ). Quantitative PCR was performed using SYBR®Premix Ex TaqTM II (Tli RNaseH Plus) on ABI7300 (PE Applied Biosystems, Waltham, MA, USA) and determined by the 2−ΔΔCT method [22 (link)].
Sybr premix ex taqtm 2 tli rnaseh plus
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR®Premix Ex TaqTM II (Tli RNaseH Plus) is a ready-to-use, high-performance PCR master mix designed for real-time quantitative PCR (qPCR) analysis. It contains SYBR Green I dye, Tli RNase H Plus, and optimized buffer components for efficient and sensitive detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Abi 7300, by Thermo Fisher Scientific (2 mentions)
Perfect real time, by Takara Bio (1 mentions)
Abi 7300 fast real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
5 protocols using sybr premix ex taqtm 2 tli rnaseh plus
1
RT-qPCR Validation of RNA-seq Data
2
Quantitative Real-Time PCR Transcriptome Analysis
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Quantitative Real-Time PCR (qPCR) was performed as described previously (17 (link)). Total RNA was obtained from the hepatic samples using Trizol Reagent (TaKaRa, Dalian, China) and then reverse-transcribed using a commercial kit (Perfect Real Time, SYBR@ PrimeScriptTM, TaKaRa) following the manufacturer's instructions. The mRNA expression levels of specific genes were quantified via real-time PCR using SYBR@Premix Ex TaqTM II (Tli RNaseH Plus) and an ABI 7300 Fast Real-Time PCR detection system (Applied Biosystems, Foster City, CA). The SYBR Green PCR reaction mixture consisted of 10 μl SYBR@Premix Ex Taq (2X), 0.4 μl of the forward and reverse primers, 0.4 μl of ROX reference dye (50X), 6.8 μl of ddH2O, and 2 μl of cDNA template. Each sample was amplified in triplicate. The fold-expression of each gene was calculated according to the 2−ΔΔCt method (17 (link)), and the β-actin gene was used as an internal standard. The primer sequences that were used are listed in Supplementary Table S1 .
3
Verification of RNA-seq by RT-qPCR
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A total of 9 DEGs were selected to verify the RNA-seq results by RT-qPCR. The specific primers of reference [24 (link)] and primers sequences used for RT-qPCR (https://www.ncbi.nlm.nih.gov/ ) were synthesized by Sangon Biotech (seen in Table 1 ). Quantitative PCR was performed using SYBR®Premix Ex TaqTM II (Tli RNaseH Plus) on ABI7300 (PE Applied Biosystems, USA) and determined by the comparative CT method [25 (link)]. The PCR cycling conditions were followed as: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing and elongation at 60 °C for 31 s, and 40 cycles for each PCR program.
4
Quantitative PCR Analysis of Porcine Viral Antigen-Stimulated Splenocytes
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Splenocytes were placed into a 24-well plate at 1 × 107 cells/well and then restimulated with inactivated PrV antigen (5 × 105 TCID50) at 37°C in 5% CO2 atmosphere for 15 h. Cells were collected after centrifugation (1500 rpm, 4°C, 5 min) and washed with ice-cold PBS. EASY spin Total RNA Extraction Kit (TaKaRa, Dalinan, China) was used to isolate total RNA. Then, PrimeScript™ RT reagent kit (TaKaRa, Dalinan, China) was used to convert total RNA into cDNA. The primers, synthesized by Sangon Biotech, met the NCBI/Primer-BLAST standards and their sequences are listed in Table 1 . Quantitative PCR was performed using SYBR Premix Ex TaqTM II (Tli RNaseH Plus) on ABI7300 (PE Applied Biosystems, USA) and data were determined using comparative CT method (2-△△CT) [33 (link)–35 (link)], where CT means the fractional cycle number at which the amount of amplified target reaches a fixed threshold, and formula −△△CT = −(△CT·Target − △CT·Control), which the relative levels of the target gene mean the fold change of target gene expressions related to the untreated control [36 (link)].
5
Quantitative Real-Time PCR Analysis
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Total RNA samples were obtained from HepG2 cells and liver tissues using RNAiso Plus reagent following the manufacturer's protocol. After purity determination, cDNA was synthesized using a PrimeScript® RT reagent Kit with a TC-512 PCR system (TECHNE, United Kingdom). The levels of mRNA were performed by using real-time PCR with SYBR® PremixEx Taq TM II (Tli RNaseH Plus) in an ABI 7500 Real Time PCR System (Applied Biosystems, United States). The forward (F) and reverse (R) primers for the tested genes are listed in Table 1 . Among the data from each sample, the Ct value of the target genes was normalized to that of GAPDH. The relative quantification of mRNAs was calculated using the 2−△△Ct method.
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