Thermo scientific revertaid first strand cdna synthesis kit

Total RNA was extracted using Invitrogen AmbionTRIzol® LS reagent
(Invitrogen Life Technologies) and reverse transcribed using Thermo Scientific
Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA,
U.S.A.) according to the manufacturer's instructions. qRT-PCR was carried
out with FastStart Universal SYBR Green Master (Rox) (Roche FastStart Universal
SYBR Green Master (Rox) (Roche Applied Science, Mannheim, Germany) on an ABI
Prism 7900 Fast instrument (Applied Biosystems, Foster City, CA, U.S.A.).
Reactions were performed in triplicate.
Primers for qRT-PCR reactions were as follows: human IL-6R, forward
5′-TTCTACAGACTACGGTTTGAG-3′ and reverse
5′-GGATGACACAGTGATGCT-3′; human IL-6ST, forward
5′-ACTGTTGATTATTCTACTGTGTAT-3′ and reverse
5′-AATTATGTGGCGGATTGG-3′; human BCL2, forward
5′-GATGACTGAGTACCTGAACC-3′ and reverse
5′-AGTTCCACAAAGGCATCC-3′; human CCND1, forward
5′-CGGAGGAGAACAAACAGA-3′ and reverse
5′-GCGGATTGGAAATGAACTT-3′; human MCL1, forward
5′-CAGGATTGTGACTCTCATT-3′ and reverse
5′-CCTCTACATGGAAGAACTC-3′; human MMP2, forward
5′-ACAAGAACCAGATCACATACAG-3′ and reverse
5′-TCACATCGCTCCAGACTT-3′; human GAPDH, forward
5′-GTCAAGGATTTGGTCGTATT-3′ and reverse
5′-AGTCTTCTGGGTGGCAGTGAT-3′. Then, the mRNA level of
IL-6R, IL-6ST, BCL2, CCND1, MCL1, and MMP2genes was normalized to GAPDH mRNA and expressed by
2^−ΔΔC_T(ΔΔC_t, the relative cyclic value)
[21 (link)].

To validate the DE circRNAs identified by RNA-seq was conducted using qRT-PCR analyses of 64 total RNA samples. A Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) was used to synthesize cDNA templates from 2 μg of total RNA. The RT-PCR amplification was performed on the ABI 7500 real-time PCR system (Applied Biosystems, CA, USA) by using TB Green™ Premix Ex Taq™ II (TaKaRa, Dalian, China). The optimum reaction conditions were as follows: 95 °C for 30 secs for predenaturation, followed by 40 cycles of 95 °C for 5 secs and 60 °C for 30 secs. The melting curves exhibited a single peak, indicating the specificity of the amplification systems and analysis of relative gene expression. All primers for circRNAs were designed and synthesized by Sangon Biotech (Shanghai, China). GAPDH was used as the internal control. The RT-PCR results were analyzed by using the 2^−∆∆Ct method.

The TIANamp Genomic DNA Kit (TIAN GEN DP304) was used for DNA extraction. Total RNA from Vero or LLC-PK1 cells was extracted using TRIzol (Vazyme), and 1 μg cDNA was synthesized using the Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. The SYBR Green master mix (Vazyme) was used to quantify the abundance of the target mRNA according to the manufacturer’s instructions. The procedure for real-time PCR consisted of an initial step at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 95 °C for 15 s. Specific primers are shown in Table 1. The PRV viral gB gene level was determined by absolute quantification real-time PCR. Nectin-1, nectin-2, and CD155, which are believed to be the PRV receptors, were quantified by quantitative PCR. The relative level of RNA expression was determined by the 2^−ΔΔCT method. β-actin mRNA level was used as a loading control. The mean RNA level of the negative control (NC) group was set at 100%.

After a cycle of drug administration in all groups (sham, MCAO model, low-ZXC, and high-ZXC; three rats/group/detection time point), the MCAO operation was performed on the rats in the MCAO model, low-ZXC, and high-ZXC groups. All rats were anesthetized using 10% chloral hydrate. For the sham group, no sutures were made, and a right-side cerebral artery occlusion model was prepared for the evaluation. After the MCAO operation, decapitation was performed at of I-30, I-90, I-90 + R-30, and I-90 + R-180. RNA Lyzol (Shanghai ExCell Biology, Inc., Shanghai, China) was used for RNA extraction. A Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for reverse transcription, and 5 μg of RNA was used for each sample. RT-PCR Master Mix (Toyobo Co., Ltd., Osaka, Japan) was used for RT-PCR, which was run on an ABI 7500 fast real-time fluorogenic quantitative RT-PCR system.

HEB, SHG44, and A172 glioma cells were provided by Xiangya Medical School of Central South University, Changsha, China. HEB, A172, and SHG44 cells were cultured in DMEM high glucose medium (Gibco/Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum at 37°C, 5%CO₂. SiRNAs were transfected with Lipofectamine 2000 (Thermo Fisher Scientific) 48 h before analysis. The siRNAs against the ETV2 gene were synthesized by RiboBio Corporation (Product number: siG000002116A-1-5, Guangzhou, China). Total RNA from HEB, SHG44, and A172 cells was extracted by the Trizol lysis method. cDNA synthesis was performed according to the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). The RNA levels of ETV2 were detected by using real-time quantitative PCR (qRT-PCR) according to the manufacturer’s protocol. The expression of ETV2 and GAPDH was analyzed by the 2^-ΔΔCt method. The primers were obtained from Sangon (Shanghai, China) and the sequences were designed as follows: for ETV2, the forward primer was 5'-CTGGAAAGGTACAAGCTCATCC-3' and the reverse primer was 5'-AACTTCTGGGTGCAGTAACGC-3'. For GAPDH, the forward primer was 5'-CATTGACCTCAACTACATGGTT-3' and the reverse primer was 5'-CCATTGATGACAAGCTTCCC-3'.

In this experiment, 56 pairs of primers of the genes showing different expression levels in our “omics” analysis or are known genes associated with chalkiness were designed and used for qRT-PCR analysis (Supplementary Tables 7, 8). Total RNA was isolated from dehulled kernels with TaKaRa MiniBEST Universal RNA Extraction kits (Takara Bio Inc., China). cDNA synthesis was carried out with the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States). The actin gene (LOC_Os03g50885) was used as the reference expression gene. SYBR Green II real-time PCR was carried out using the TransStart^® Top Green qPCR Super Mix Kit (TransGen Biotech, China) on an ABI Prism 7500 Sequence Detector (Applied Biosystems, Inc., United States). The real-time PCR amplification mixture (20 μl) contained 1 μg of cDNA, 10 μl of 2 × TransStart^® Top Green qPCR Super Mix Kit, 0.4 μl of 50 × Dye II and 4 μl of 5 μM forward and reverse primers.

Total RNA was extracted from different tissues and primary hepatocyte using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Each of the RNA samples was treated with DNase I (Invitrogen #18068015) to remove trace amounts of genomic DNA according to the manufacturer’s instructions. The integrity of total RNA was confirmed by gel electrophoresis, and the RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The RNA samples with OD260/280 ratios above 1.8–2.0 were used for further study. Single-stranded cDNA was synthesized using random hexamer primers with the Thermo Scientific™ Revert Aid First Strand cDNA Synthesis kit (Thermo Scientific) following the manufacturer’s protocol. The cDNA was stored at −20 °C until use.