For quantitative real-time RT-PCR, the reference genes 18S (GenBank No. AY049040) and actin (GenBank No. AB181911) (Yamada et al., 2009; Wang et al., 2010) were used (Table 1); the PrimeScript Perfect Real-Time RT Reagent Kit (TaKaRa, China) was used for the reactions. The primers were designed using the Primer Express 3.0 software for the HGF2, HDB2, and HCG4 genes (Table 1). PCR was conducted in triplicate and normalized to 18S and actin. Real-time expression assays were performed with SYBR Green dye (TaKaRa) using the BIO-RAD CFX-96 real-time PCR platform (Bio-Rad). Fold-changes of RNA transcripts were calculated via the 2 -ΔΔCt method (Livak and Schmittgen, 2001) .
Primescript rt reagent kit perfect real time
Manufactured by Takara Bio
Sourced in Japan, China
The PrimeScript RT Reagent Kit Perfect Real Time is a laboratory equipment product designed for reverse transcription (RT) reactions. It provides the necessary reagents and enzymes required for the conversion of RNA into complementary DNA (cDNA) for subsequent real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (42 mentions)
Sybr premix ex taq 2 tli rnaseh plus, by Takara Bio (13 mentions)
Lightcycler 480 2 real time pcr system, by Roche (11 mentions)
Sybr premix ex taq, by Takara Bio (10 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (6 mentions)
Sybr premix ex taq kit, by Takara Bio (5 mentions)
Lightcycler 480 sybr green 1 master, by Roche (5 mentions)
Sybr premix ex taqtm 2, by Takara Bio (5 mentions)
Sybr premix ex taq 2, by Takara Bio (5 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (5 mentions)
High pure rna isolation kit, by Roche (5 mentions)
Rnaiso plus, by Takara Bio (5 mentions)
Cfx96 real time system, by Bio-Rad (4 mentions)
Rneasy micro kit, by Qiagen (4 mentions)
Trizol reagent, by Takara Bio (4 mentions)
Sybr premix ex taqtm 2 tli rnaseh plus, by Takara Bio (4 mentions)
Sybr green real time pcr master mix kit, by Toyobo (3 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
170 protocols using primescript rt reagent kit perfect real time
1
Quantitative Real-Time RT-PCR Analysis
2
Quantifying Wheat WAG-1 Expression
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Total RNA was isolated from spikes of the sister wheat lines CSTP and HTS-1 at various development stages (2-5, 5-7, 7-10 mm in length). cDNA was synthesized using a PrimeScript Perfect real-time RT reagent kit (TaKaRa, Dalian, China), following the manufacturer protocol. The primers for WAG-1-Q (Table 1) were designed using Prime Primer Premier 5.0 software to amplify short fragments of WAG-1. Real-time assays were performed with SYBR green (TaKaRa) using a Bio-Rad CFX96 real-time PCR platform. All samples were analyzed in triplicate and the fold change in the number of RNA transcripts was calculated using the 2 -ΔΔCt method (Livak and Schmittgen, 2001) with the wheat housekeeping genes ubiquitin (DQ086482) and actin (AB181911) acting as internal controls (Hama et al., 2004; Yamada et al., 2009) .
3
Quantification of DlRan3A and DlRan3B Gene Expression in Longan
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Total RNA was extracted using the RNAprep Pure Plant Kit (TIANGEN Code, DP441, Beijing, China) or total plant RNA extraction kit (BioTeke Code, RP3312, Beijing, China) following the manufacturer’s protocol. cDNAs were synthesized using the PrimeScriptTM Perfect Real-Time RT Reagent Kit (TaKaRa Code, RR037A (Dalian, China)). Quantitative real-time PCR analysis (qPCR) was performed to evaluate the transcript levels of the DlRan3A and DlRan3B genes in longan tissues, during zygotic embryo and pulp developments. Typical reactions were prepared using the SYBR Premix Ex Taq kit (Takara) and all the qPCR reactions were performed in triplicate. QPCR assays were implemented using the LightCycler 480 qPCR instrument (Roche Applied Science, Basel, Switzerland) and cycling conditions were chosen according to the manufacturer’s protocol. The expression profiles of DlRan3A and DlRan3B in longan tissues were quantified using three pre-microRNAs (pre-miR167f3p, pre-miR171f, and pre-miR394a) as the reference genes; and the expression levels during zygotic embryo development were quantified using the 2−ΔΔCT method, with longan Fe-SOD as a reference gene. The expression levels of DlRan3A and DlRan3B in other longan samples were quantified using the internal standards as described previously [53 (link)]. Primer names and sequences are provided in Table S1 .
4
Quantitative Analysis of miRNA and mRNA
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Total RNAs were isolated from the nine synchronized embryogenic cultures described above using the TRIzol Reagent kit (Invitrogen). cDNAs for miRNA and mRNA quantification were synthesized using a One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara Code, D350A), and PrimeScriptTM Perfect Real Time RT Reagent Kit (TaKaRa Code, DR037A), respectively.
Quantitative PCR assays of gene expression were performed using a LightCycler 480 qPCR instrument (Roche Applied Science, Switzerland) and SYBR premix (Takara). All reactions were performed in triplicate. The expression levels of the miR160s, their targets (ARF10, -16, -17), and the eTMs were quantified using internal standards (Lin and Lai, 2010 (link), 2013c (link)). Statistical analysis was performed using SPSS 19. Gene names and primer sequences are provided inTable 2 .
Quantitative PCR assays of gene expression were performed using a LightCycler 480 qPCR instrument (Roche Applied Science, Switzerland) and SYBR premix (Takara). All reactions were performed in triplicate. The expression levels of the miR160s, their targets (ARF10, -16, -17), and the eTMs were quantified using internal standards (Lin and Lai, 2010 (link), 2013c (link)). Statistical analysis was performed using SPSS 19. Gene names and primer sequences are provided in
5
RNA Extraction and qPCR Analysis in PK-15 Cells and Mice
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Total RNA was isolated from PK‐15 cells and mouse tissue using the Takara MiniBEST Universal RNA Extraction Kit (Takara), according to the manufacturer's instructions. The RNA samples were then reverse‐transcribed to cDNA using the Primescript RT Reagent Perfect Real‐Time kit (Takara). The PCR primers were designed using Primer Premier 5.0 software, and primers for GAPDH, IL‐1β, IL‐6, IL‐8 and TNF‐α are shown in Table 1 . The PCR reaction system consisted of 2 μl cDNA, 10 μl SYBR Premix Ex Taq II, 0.8 μl forward primer, 0.8 μl reverse primer and 6.4 μl ddH2O. qPCR was performed using the following program: 95°C for 15 s, 40 cycles at 95°C for 5 s and 60°C for 30 s followed by the melting curve and plate reading stages, under default settings. The relative expression level of the target genes was calculated using the comparative Ct method. All data were normalised relative to GAPDH. qPCR was performed using the Bio‐Rad CFX Connect Real‐Time System.
2.9 Animals Forty‐five seven‐week‐old female BALB/c mice were purchased from the Wuhan Institute of Biological Products Co., LTD. Mouse studies were performed according to the guidelines of this company (No. 00281346). All animal studies were complied with the Hubei Provincial Animal Care and Use Committee and the animal experiment guidelines of the Animal Experimentation Ethics Committee of Huazhong Agricultural University.
2.9 Animals Forty‐five seven‐week‐old female BALB/c mice were purchased from the Wuhan Institute of Biological Products Co., LTD. Mouse studies were performed according to the guidelines of this company (No. 00281346). All animal studies were complied with the Hubei Provincial Animal Care and Use Committee and the animal experiment guidelines of the Animal Experimentation Ethics Committee of Huazhong Agricultural University.
6
Quantitative Real-Time PCR Gene Expression
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Total RNA was extracted from cells using TRIzol (Invitrogen, USA). The total RNA concentration and purity were quantified using a spectrophotometer (Eppendorf, Germany). Reverse transcription was performed at 37 °C for 15 min followed by 85 °C for 10 s using a Prime Script RT reagent Perfect Real Time Kit (Takara Bio Inc, Japan). Quantitative PCR was performed in duplicate on a Light Cycler 480II real-time PCR system (Roche, Switzerland) with a SYBR Green Real-time PCR Master Mix kit (Toyobo, Japan). The PCR contained 3.2 μL of ddH2O, 5 μL of 2×SYBR Green Real-time PCR Master Mix, 0.4 μL of forward and reverse primers, and 1 μL of cDNA in a final volume of 10 μL. PCR conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 1 min. The primer sequences for the genes are shown in Supplementary Table 2 . To determine the specificity of the PCRs, melting curves were routinely analysed. All experiments were conducted according to the manual’s instructions. Relative gene expression was expressed relative to that of the endogenous control GAPDH and calculated using the 2–ΔCT method.
7
Quantification of lncRNA Expression
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The same total RNA used for sequencing was reverse transcribed into cDNA using the PrimeScript™ RT reagent (Perfect Real Time) kit (TaKaRa, Shiga, Japan) for quantitative real-time polymerase chain reaction analysis (qRT-PCR), which was performed on a LightCycler 480 (Roche, Rotkreuz, CH) with Actin (Achn10718) as the internal reference gene. The relative expression levels of selected lncRNAs and genes were calculated using the 2−∆∆Ct method. The information regarding primers used in this study are shown in Supplementary Materials Table S11 .
8
Quantifying PD-L1 Expression in T24 and SV-HUC-1 Cells
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Total RNA (500 ng) was extracted from cultured T24 and SV-HUC-1 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Reverse transcription was performed using the PrimeScript RT reagent (Perfect Real Time) kit (Takara Biotechnology Co., Ltd., Dalian, China). RT-qPCR was performed using the Roche capillary-based Light Cycler 2.0 system (Roche Diagnostics, Indianapolis, IN, USA) and the SYBR Premix Ex Taq™ (Perfect Real Time) kit (Takara Biotechnology Co., Ltd.) under standard thermocycling conditions (start at 95°C for 15 min and 40 cycles of 95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec). PD-L1-specific TaqMan probes and primers were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Appropriate dilutions (1:1,000) of single strand cDNA were prepared for subsequent PCR using β-actin as the quantitative control. Primers were as follows: PD-L1 forward, 5′-CACTCATCATTGGCTTTGGTATTTCAG-3′ and reverse, 5′-CGACAGCTCATCTTTGCCTTCTTTG-3′; and β-actin forward, 5′-AGCGGGAAATCGTGCGTGAC-3′ and reverse, 5′-ACTCCTGCTTGCTGATCCACATC-3′. Each experiment was performed in triplicate and analyzed using the 2−∆∆Cq method of quantification (13 (link)).
9
RT-qPCR Validation of DEGs
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Eight DEGs that were significantly differentially expressed in at least one isolate-infected group in each tissue were selected for the quantitative reverse transcript PCR (RT-qPCR) validation. As mentioned above, total RNA was extracted from spleen and liver tissue samples. The manufacturer’s instructions were followed to produce cDNA from the total RNA using a PrimeScript RT reagent Perfect Real Time Kit (TaKaRa, Dalian, China). Then, the reaction of qPCR was performed and analysed using a Rotor-Gene Q Series Software 1.7 supplied with the instrument (QIAGEN, Hilden, German). An amount of 10 μL of TB Green Premix Ex Taq Ⅱ (TaKaRa, Dalian, China), 2 μL of the cDNA sample, 0.8 μL (10 μM) of each primer, and ddH2O in a total volume of 20 μL made up the reaction mixtures. The reactions were amplified for 30 s at 95 °C, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s.
Primer sequences of eight DEGs were listed inTable 1 . For normalization of gene expression, β-actin gene was used as an internal control. Primers had a Tm of roughly 60 °C, and PCR products ranged in length from 100 to 200 bp. qPCR was conducted three times for each sample as technique replicates.
Primer sequences of eight DEGs were listed in
10
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from sporophytes and gametophytes using the Spectrum Plant Total RNA Kit (Sigma). After RNA extraction, the samples were submitted to a DNase I treatment (REAL Star Kit, Durviz, Paterna, Spain), to ensure the absence of contaminating genomic DNA traces. RNA quantity and purity were measured using Tecan’sInfinite 200 NanoQuant. For each sample, 100 ng RNA was retrotranscribed into complementary DNA (cDNA) using the combination of random and oligo deoxythymidine (dT) primers of the PrimeScript RT reagent—Perfect Real Time-Kit (Takara) and following manufacturer’s recommendations. For each analysis, a negative control devoid of reverse transcriptase was included to test for contaminating DNA.
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