The abundance of the amoA gene and gene transcripts was determined by the StepOnePlus quantitative PCR (qPCR) system (Applied Biosystems, Inc., Carlsbad, CA, United States), with 25 μl of the SYBR® Premix Ex TaqTM kit (Takara Bio, Inc., Shiga, Japan), 0.3 μM of the Arch-amoAF/Arch-amoAR primer (Francis et al., 2005 (link)) and 2 μl of each DNA/cDNA as the template. The standard curve for absolute quantification was constructed using plasmid amplicons that were quantified on an Agilent 2100 bioanalyzer using DNA 7500 chips, according to the manufacturer’s protocol (Agilent Technologies, Inc., Santa Clara, CA, United States). Triplicate qPCR reactions were performed for each sample with efficiencies around 110%, and the gene copy number was normalized to the quantity of the gene and gene transcripts. The theoretical copy number was calculated to the size of the input PCR amplicon. In parallel, negative controls without reverse transcriptase and template were also prepared for the cDNA samples and no amplicons were produced. In addition, AOA group-specific assays for “shallow” water column ecotype A (WCA) and “deep” water column ecotype B (WCB) (Mosier and Francis, 2011 (link)) were conducted with efficiencies around 93% using the SYBR® Premix Ex TaqTM kit (Takara Bio, Inc.) (Santoro et al., 2013 (link)).
Sybr premix ex taqtm kit
Manufactured by Takara Bio
Sourced in Japan, China, United States, Switzerland, Germany, Spain, Australia, Canada
The SYBR Premix Ex Taq™ kit is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR® Green I dye, Taq DNA polymerase, and other necessary reagents. The kit is designed for sensitive and reliable quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (128 mentions)
Primescript rt reagent kit, by Takara Bio (63 mentions)
Trizol, by Thermo Fisher Scientific (25 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (24 mentions)
Trizol reagent, by Takara Bio (23 mentions)
Primescript rt master mix, by Takara Bio (21 mentions)
Lightcycler 480, by Roche (16 mentions)
Trizol kit, by Thermo Fisher Scientific (14 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (13 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
Primescript rt master mix kit, by Takara Bio (12 mentions)
Rnaiso plus, by Takara Bio (11 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (10 mentions)
Cfx96 real time system, by Bio-Rad (10 mentions)
Lightcycler 480 2 system, by Roche (9 mentions)
Primescript rt kit, by Takara Bio (9 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (8 mentions)
Rnaiso plus reagent, by Takara Bio (7 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (7 mentions)
418 protocols using sybr premix ex taqtm kit
1
Quantitative Analysis of Archaeal amoA Gene
2
Quantitative Gene Expression Analysis
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Total RNA was extracted using RNA Prep Pure Plant kit (TIANGEN) and reverse-transcribed with a FastKing RT Kit (TIANGEN) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using a SYBR_ Premix Ex TaqTM kit (TaKaRa) on an ABI Q3 Real-Time PCR System. Relative gene expression was analyzed using 2-ΔΔCT method (Livak and Schmittgen 2001 (link)). Primers for quantitative Real-Time PCR were designed by Primer Premier 5.0 or GenScript. The ubiquitin gene (ubq) was used as a reference (Suppl. Table S1 ).
3
Quantitative Expression Analysis of Photosynthesis Genes in Rice
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Total RNA was extracted from seedling roots, young stems, young leaves, flag leaves and young panicles using an RNA Prep Pure Plant kit (Tiangen Co., Beijing, China). For RT-PCR, first-strand cDNA was reverse transcribed from total RNA with RT primer mix (oligo dT and random 6 mers). Real-time PCR was performed using a SYBR_ Premix Ex TaqTM kit (TaKaRa) on an ABI prism 7900 Real-Time PCR System. The 2-ΔΔCT method was used to analyze the relative changes in gene expression (Livak and Schmittgen 2001 (link)). The primers for photosynthesis and chloroplast development associated genes (FtsZ, OsRpoTp, rpoB, rpoC1, rpoC2, Cab1R, rbcS, RbcL, psbA, 16S rRNA, 23S rRNA) were listed in Additional file 2 : Table S2. The rice Actin gene was used as a reference gene in this study.
4
Quantification of Soil Nitrification Genes
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Soil DNA was extracted from 0.25 g fresh soil using MoBio PowerSoil DNA isolation Kit (MoBio Laboratory, Carlsbad, CA, United States). The quality and integrity of total DNA were assessed by 1% agarose gel electrophoresis. Quantification of functional marker genes (AOA, AOB, and Comammox Nitrospira clade A amoA, Nitrobacter-like nxrA, and Nitrospira-like nxrB) was carried out by quantitative PCR using a CFX96 Optical Real-Time Detection System (Bio-Rad, United States). The primer pairs and amplification procedures for quantification of functional genes are listed in Supplementary Table 1 . The SYBR Premix Ex TaqTM Kit (TaKaRa Biotechnology Co., Dalian, China) was used to conduct quantitative PCR reactions. The PCR reaction (25 μl total) contained 12.5 μl 2 × SYBR Premix Ex TaqTM (Takara, Dalian), 0.5 μl of each primer, 2 μl DNA template (1–10 ng), and 9.5 μl dd H2O. Standard curves were obtained using plasmid DNA containing right inserts of the functional genes. Specific amplification was checked through a melting curve and a 1.5% agarose gel. However, we failed to amplify the band of Comammox Nitrospira clade B according to the gel electrophoresis detection system, and thus Comammox Nitrospira clade B was excluded from further analyses. The amplification efficiencies resulted in values of 88–95% for all functional genes, and the r2 values were > 0.99.
5
Quantitative Analysis of miRNA and mRNA
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Total RNA was extracted from cells according to the instructions of the MiRcute
miRNA Extraction and Separation Kit (Tiangen, Beijing). The concentration and
purity of RNA were measured by a microspectrophotometer (BioDrop, UK). BRAF and
miR-9-5p cDNA were synthesized with Prime Script RT Master Mix (Takara, Dalian)
and Mir-XTM miRNA First-Strand Synthesis Kit (Takara, Dalian),
respectively. The real-time PCR primer sequences are displayed inTable 1 . To measure
the relative levels of both miRNA and mRNA, a LightCycler 480 (Roche, Swiss) was
used for PCR with a SYBR Premix Ex TaqTM kit (Takara, Dalian). Each
sample was assayed 3 times. The experimental results were quantitatively
analyzed by the 2-ΔΔCt method.
miRNA Extraction and Separation Kit (Tiangen, Beijing). The concentration and
purity of RNA were measured by a microspectrophotometer (BioDrop, UK). BRAF and
miR-9-5p cDNA were synthesized with Prime Script RT Master Mix (Takara, Dalian)
and Mir-XTM miRNA First-Strand Synthesis Kit (Takara, Dalian),
respectively. The real-time PCR primer sequences are displayed in
the relative levels of both miRNA and mRNA, a LightCycler 480 (Roche, Swiss) was
used for PCR with a SYBR Premix Ex TaqTM kit (Takara, Dalian). Each
sample was assayed 3 times. The experimental results were quantitatively
analyzed by the 2-ΔΔCt method.
6
Transcriptome Analysis Using RNA Extraction and qPCR
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Nuclear and cytoplasmic RNA was extracted according to the instruction of RNA Subcellular Isolation Kit (Active Motif, Carlsbad, CA, USA). Total RNA from tissues and cell lines was isolated using RNeasy Mini Kit (QIAGEN, Germany) following the manufacturer’s instructions. Reverse transcription and real-time PCR were performed using reverse transcription kit PrimeScript RT Master Mix (Takara, Dalian, China), the SYBR Premix Ex TaqTM kit (Takara), and primers shown in Table S1 . The results of transcript levels were analyzed by 2−ΔΔCt method.
7
Quantitative RT-PCR Using SYBR Premix
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qRT-PCR was performed in 96-well blocks with a PikoReal Real-time PCR System (Thermo Fisher Scientific) using the SYBR Premix Ex TaqTM Kit (TaKaRa Biotechnology). Reactions were performed with 2 of diluted cDNA, 200 nM of each primer (Invitrogen) and 10 μL of 2 × SYBR PCR Master Mix with the following amplification conditions: 95 °C for 10 min, and 45 cycles of 95 °C for 15 s and 60 °C for 1 min. All measurements were performed using three biological replicates. Negative RT control, negative poly (A) polymerase controls, and non-template controls were also included in each run.
8
Quantifying Bacterial Biomass via qPCR
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qPCR assays were conducted to determine total bacterial biomass using the SYBR Premix Ex TaqTM Kit (Takara, Beijing, China) and iQ5 Real-Time PCR System (Bio–Rad, Hercules, CA, USA). Universal primers Eub338/Eub518 [29 (link)] were used to identify the V4 regions in the bacterial 16S rRNA genes. The PCR mix contained ten microliters each of SYBR green I and ROX dye II (Applied Biosystems, Waltham, MA, USA), 0.4 microliters of each primer, 2.0 microliters of DNA, and sterile deionized water up to a total of twenty microliters. The qPCR conditions entailed initial denaturation at 95 °C for 5 min, followed by 37 cycles of denaturation at 95 °C for 45 s, annealing at 56 °C for 45 s, and a final extension at 72 °C for 2 min. The standard samples were diluted to conduct a set of 10-fold dilutions and then used to quantify gene expression with qPCR. The standard curves were generated according to a predetermined protocol [29 (link)], and the R2-value exceeded 0.99. All amplifications from each individual sample were measured in triplicate.
9
Primer Design and qRT-PCR Analysis of CsTCP Genes
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The specific primer design of each CsTCP gene was implemented on an online Primer3 website (http://primer3.ut.ee ), and are shown in Table S3 . The reference gene sequences that quantitative real-time (qRT-PCR) used are 5′-GGCAGTGGTGGTGAACATG-3′ and 5′-TTCTGGTGATGGTGTGAGTC-3′. The qRT-PCR setting to three replications was performed using an SYBR Premix Ex TaqTM kit (TaKaRa, Dalian, China) and a Roche LightCycler480 System (Bio-Rad, Hercules, CA, USA). Relative expression levels of CsTCPs were calculated with the comparative threshold method (2−ΔΔCt) and shown on a heatmap using HemI 1.0 software. The expression of CsTCPs under WMV stress was analyzed by real-time quantitative PCR (qRT-PCR) technology; treatments covered 1, 3, 6, 9, 12, and 18 days after inoculation.
10
Quantifying miRNA and Gene Expression
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Total RNA was extracted and reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara, Japan). qRT-PCR was performed using the SYBR Premix Ex TaqTM kit (Takara, Japan). The expression levels of miR-22, ErbB3, and Nr3c1 relative to the expression level of GAPDH were determined using the 2-ΔΔCT method. The specific primer sequences are as follows: ErbB3 (forward), 5’-TCTGCATTAAAGTCATCGAGGAC-3’ and ErbB3 (reverse), 5’-CAGCCGTACAATGTGGGCAT-3’; Nr3c1 (forward), 5’-CTTGAGAAACTTACACCTCGATGACC-3’ and Nr3c1 (reverse), 5’-AGCAGTAGGTAAGGAGATTCTCAACC-3’; and GAPDH (forward), 5’-TCTCTGCTCCTCCCTGTTCT-3’ and GAPDH (reverse), 5’-TACGGCCAAATCCGTTCACA-3’. The miR-22 primer was designed and synthesized by RiboBio (Guangzhou, China).
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