Total RNA was extracted from tomato leaves using an E.Z.N.A.® Plant RNA Kit (Omega Bio–Tek, Doraville, GA, USA) according to the manufacturer’s instructions. The total RNA was then reverse–transcribed using a PrimeScriptTM RT reagent kit with gDNA Eraser (Takara, Shiga, Japan) in a 20–μL reaction mixture containing 1 μL of total RNA from each individual sample. Real–time PCR was performed on a CFX96™ real–time PCR cycler (Bio–Rad, Hercules, CA, USA) and a SYBR Premix Ex Taq (TliRNaseH Plus) Kit (Takara). Initial denaturation at 95 °C for 30 s was followed by 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and a melting curve of 65–95 °C. Primers for the actin gene were used as an internal control. Primers for psbA and actin were designed as described by Wu et al. [55 (link)]. Primers for the pbgD and Chlase genes were designed using Primer3, version 4.0.0 (website software), with the primer length set at 20 − 24 bp; melting temperature of 58 − 62 °C; CG content, 30 − 70 %; and product size, 150–250 bp. All samples were analyzed three times.
Sybr premix ex taq tli rnaseh plus kit
Manufactured by Takara Bio
Sourced in Japan, United States, China, France, Germany
The SYBR Premix Ex Taq (Tli RNaseH Plus) kit is a real-time PCR reagent that includes a hot-start DNA polymerase, dNTPs, SYBR Green I dye, and buffer components. It is designed for accurate and sensitive quantification of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (7 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (7 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Primescript rt master mix kit, by Takara Bio (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Sybr primescript mirna rt pcr kit, by Takara Bio (2 mentions)
Abi7500 quantitative pcr instrument, by Thermo Fisher Scientific (2 mentions)
Abi 7500 quantitative pcr machine, by Thermo Fisher Scientific (2 mentions)
Abi 7500 qpcr instrument, by Thermo Fisher Scientific (2 mentions)
Trizol, by Takara Bio (2 mentions)
Cfx96 real time pcr system, by Bio-Rad (2 mentions)
Primescript rt kit, by Takara Bio (2 mentions)
Rnaiso plus reagent, by Takara Bio (2 mentions)
Minibest universal rna extraction kit, by Takara Bio (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
53 protocols using sybr premix ex taq tli rnaseh plus kit
1
Tomato Leaf RNA Extraction and qRT-PCR
2
Quantitative Analysis of Nitrogen Fixation Genes
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For quantitative real-time-PCR (qRT-PCR), cultures of P. polymyxa WLY78, ∆nifS-like, ∆yutI, ∆sufCDB, ∆sufC, and ∆sufD were grown under N2-fixing conditions (2 mM glutamate and without O2) and harvested after 8 h of incubation. To detect the expression of the nifU gene, total RNA was extracted from ∆sufC, ∆sufD, ∆sufB, ∆sufCDB, ∆sufC/nifU-K.o, ∆sufD/nifU-K.o, ∆sufB/nifU-K.o, and ∆sufCDB/nifU-K.o. Total RNA was isolated using TRIzol (Takara Bio, Tokyo, Japan). The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I (Takara Bio, Tokyo, Japan). The integrity and size distribution of the RNA were verified by agarose gel electrophoresis, and the concentrations were determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specifications (Takara Bio, Tokyo, Japan). cDNA (0.4 µg) was used for qRT-PCR. The relative transcript levels of sufD, sufS, sufU, and sufB were determined with 16S rDNA as a control by the SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara Bio, Tokyo, Japan). Primers for sufC, sufD, sufS, sufU, sufB, nifH, nifD, nifU, and 16S rDNA used for RT-PCR or qRT-PCR are listed in Table S3 .
3
Quantitative Analysis of PTTG1 mRNA and miRNAs
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Total RNA was isolated from the specimens and cell lines using TRIzol reagent (Invitrogen, USA). For PTTG1 mRNA analysis, complementary DNA was synthesized by using the reverse transcription kit (RR047A, TaKaRa). For the detection of mature miRNAs, the RNA was reversely transcribed into cDNA by using the SYBR® PrimeScript™ miRNA RT-PCR Kit (RR716, TaKaRa). SYBR® Premix Ex Taq™ (Tli RNaseH Plus) kit (RR420A, TaKaRa) was then utilized for Real-time RT-PCR detection. The nucleotide sequences of the primers were as follows: PTTG1: 5′-CCCTCAAACAAAAACAGCCAAG-3′ (forward), 5′-GGCATCATCTGAGGCAGGAAC-3′ (reverse). GAPDH served as an internal control using primers 5′-TC AAGAAGGTGGTGAAGCAG-3′ (forward) and 5′-CG TCAAAGGTGGAGGAGTG-3′ (reverse). The specific premiers for the candidate miRNAs were purchased from RiboBio (RiboBio Co., Ltd., China), and U6 was used as an internal control for detection of miRNAs. All analyses were performed by using the StepOnePlusTMReal-Time RT-PCR System (Applied Biosystems, USA). The 2−ΔΔCT method was employed to calculate the relative expression of each gene.
4
RT-qPCR Analysis of Diverse Genes
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For real-time reverse transcription–quantitative PCR (RT-qPCR) analysis, cells were lysed and total RNA was extracted using RNeasy Mini Kit including an on-column DNase digestion step (both Qiagen). RNA was reverse transcribed using random primers and the High Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer's specifications. Real-time qPCR for human YAP, endothelin-1 (EDN1), EDN2, V-myc myelocytomatosis viral oncogene homolog (avian) (MYC), cadherin-6 (CDH6), cysteine-rich, angiogenic inducer, 61 (CYR61), thrombospondin-1 (THBS1), and growth arrest and DNA damage-inducible, beta was performed using the SYBR Premix Ex Taq (Tli RNase H Plus) Kit (Takara Bio Europe, Saint-Germain-en-Laye, France) on a Reaplex2 Mastercycler Real-Time PCR System (Eppendorf, Hamburg, Germany). Relative fold expression levels were determined using the 2(− ΔΔCt) method [15] (link), with GAPDH used as a housekeeping control.
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from RAW264.7 cells with RNAiso Plus (Takara Bio, Inc., Otsu, Japan). RT was carried out using a PrimeScript RT Reagent kit (Perfect Real Time; Takara Bio, Inc.) following the manufacturer's protocol. A total of 2 µg total RNA was reverse-transcribed in a volume of 10 µl for cDNA synthesis. The resulting cDNA was amplified using a SYBR® Premix Ex Taq (Tli RNaseH Plus) kit (Takara Bio, Inc.). qPCR was performed in the CFX96™ Real-Time PCR system (Bio-Rad Laboratories, Inc.,). The PCR primers used are presented in Table I . The reaction system consisted of 2 µl sense primer, 2 µl anti-sense primer, 12.5 µl SYBR® PCR Master mix, 2 µl template cDNA (25 ng/µl) and double-distilled water to a final volume of 25 µl. PCR was performed for 40 cycles, and each cycle included denaturation at 95°C for 5 sec, annealing and extension at 60°C for 30 sec, which followed pre-denaturation at 95°C for 30 sec. The relative copy number was analyzed by using the threshold crossing point (Cq), which was calculated by the system's software, combining with the 2−ΔΔCq calculations (23 (link)).
6
RNA Extraction and RT-qPCR for Gene Expression
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Around 109 cells were harvested for each sample and washed once with pre-chilled diethyl pyrocarbonate (DEPC)-treated water. Cell pellets were snap-frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Total RNA was obtained using the Trizol reagent (Invitrogen). RNA was then reverse transcribed using the PrimeScript RT kit (Takara, RR014A). The cDNA library was used as a template in real-time PCR using the Takara SYBR Premix Ex-Taq (Tli RNase H Plus) kit (Takara, RR420A). The primers for CLN2 and ACT1 were the same as in our previous study (Zhang Z. et al., 2017 (link)).
7
Quantitative Real-time PCR Analysis
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Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript RT reagent kit (Takara). Real-time quantitative PCR was performed in triplicate using the SYBR Premix Ex Taq (Tli RNase H Plus) kit (Takara). The expression of TBP was used for the normalization of mRNA expression. All primers used for RT-qPCR were listed in Supplementary Table 3 .
8
DMEM-based Cell Culture Protocol
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Dulbecco’s modified eagle medium (DMEM) (Gibco) supplemented with streptomycin 100 IU/mL, penicillin 100 IU/mL, 10% fetal bovine serum and glutamine 0.75 mg/mL was used as the nutritive medium. The fetal bovine concentration of the maintain medium (MM) was reduced to 1 %. Dulbecco’s Hanks Balanced Salt Solution (D-Hank’s) was used for washing the embryo. The pH of D-Hank’s, DMEM and MM solutions was adjusted to 7.4 with 5.6 % NaHCO3 and stored at 4 °C. Trypsin (Amresco) was dissolved with D-Hank’s up to 0.2 %. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco) was dissolved to 5 mg/mL with phosphate-buffered saline (PBS), filtered through a 0.22 μm syringe filter, and stored at 4 °C in dark bottles. Other chemicals used in the experiment were analytical grade. RNAiso Plus Reagent (Lot no. 9108), PrimeScript RT Master Mix Kit (Lot no. RR036A) and SYBR Premix Ex Taq (Tli RNaseH Plus) Kit (Lot no. RR036A) were bought from Takara. Fluorescein isothiocyanate-labeled rabbit anti-DHAV (Lot no. orb8860) was bought from biorbyt. DHAV (LD50:5 × 10−3) strain LQ2 used in the challenge experiments was supplied by the Shandong Institute of Poultry (Shandong, China,−70 °C storage). All other chemicals are from standard commercial suppliers, having analytical grade quality.
9
Quantitative Real-Time PCR for mRNA Expression
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A 2-step method for qRT-PCR was used to determine mRNA expression level. Briefly, cDNA was synthesized using oligo (dT) primers with PrimeScript™ II 1st strand cDNA synthesis Kit (Takara Biotechnology, China) according to the manufacturer’s protocol. qRT-PCR was performed using Applied Biosystems StepOne™ Real-Time PCR System (Applied Biosystems, USA) and SYBR Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara Biotechnology, China). The primers of these detected genes were shown in Table 5 and primers for endogenous reference gene GAPDH applied a commercial product (Sangon Biotech, China). The expression levels were measured in terms of the cycle threshold (Ct) and were normalized to GAPDH expression using the 2-△△Ct method [32 (link)].
10
Quantification of Viral RNA by qPCR
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Total RNA was extracted from cell lysates using RNeasy (QIAGEN), and an aliquot of 100 ng was reverse-transcribed with PrimeScript RT Reagent Kit with gDNA Eraser (Takara) and the included primer mix. An aliquot of 10 ng cDNA was used as a template for amplifying sequences of human GAPDH, and LRP1 and VSV with corresponding QuantiTect primers (QIAGEN) and specific primers (Table S3), respectively, and the SYBR Premix Ex Taq (Tli RNaseH Plus) kit (Takara). RRN18S was amplified in a similar manner, but with 2 ng cDNA as a template. RNA levels of EMCV, LACV, RVFV, SARS-CoV-2, and SFSV were detected using specific primers and TaqMan probes (Table S3) (Bird et al, 2007 (link); Weidmann et al, 2008 (link); Qin et al, 2018 (link); Corman et al, 2020 (link)), and the Premix Ex Taq (probe qPCR) kit (Takara). All PCRs were performed in a StepOne Plus instrument (Applied Biosystems). The values obtained for each gene were normalized against GAPDH mRNA levels (or RRN18S mRNA levels in the case of the VSV RNA) using the threshold cycle (ΔΔCT) method (Livak & Schmittgen, 2001 (link)).
Table S3.
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