RNA was extracted from excised colons stored at −80°C using an RNA extraction kit (Feiluomaige, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was produced using TaKaRaTaq™ (Takara) and KOD FX (TOYOBO, Osaka, Japan) according to the manufacturers’ instructions in a PCR instrument (Mastercycler, Eppendorf). The expression levels of target genes were determined using the SYBR®Premix Ex Taq™ kit (Takara). The following cycling conditions were used: 95°C for 3 min, followed by 45 cycles of 95°C for 30 s, 58°C for 30 s, and elongation at 72°C for 30 s. All gene primers were synthesized at Qinke Biotech. Table 2
Takara taq
Manufactured by Takara Bio
Sourced in Japan, United States, China
TaKaRa Taq is a thermostable DNA polymerase derived from the bacterium Thermus aquaticus. It is designed for use in polymerase chain reaction (PCR) amplification of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taq kit, by Takara Bio (2 mentions)
Dntp mixture, by Takara Bio (2 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Kod fx, by Toyobo (1 mentions)
Mastercycler, by Eppendorf (1 mentions)
Real time pcr master mix for sybr green 1, by Biofact (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Cfx384 real time pcr system, by Bio-Rad (1 mentions)
Superscript first strand synthesis, by Thermo Fisher Scientific (1 mentions)
Mx3000p qpcr system, by Agilent Technologies (1 mentions)
Quick gdna miniprep, by Zymo Research (1 mentions)
Dynamo colorflash sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Pikoreal real time pcr system, by Thermo Fisher Scientific (1 mentions)
Epitect plus dna bisulfite kit, by Qiagen (1 mentions)
Mce 202 multina, by Shimadzu (1 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
38 protocols using takara taq
1
Transcriptional Analysis of Colon Inflammation
2
Quantitative and Semi-quantitative RT-PCR Analysis
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RT-qPCR was performed using a Bio-Rad CFX384 real-time PCR system. PCR reaction mixture (20 μl) was prepared with 20 ng of cDNA, 10 μl of 2X Real-Time PCR Master Mix For SYBR® Green I (BioFACT, Korea), and 10 pmoles of each forward and reverse primer. The reaction condition was as follows: 15 min at 95 °C and 40 cycles of 20 s at 95 °C, 30 s at 57 °C, and 20 s at 72 °C, and the melting curve was generated by raising the temperature from 55 to 95 °C. Two reference genes, EF1α (FGSG_08811) and UBH (FGSG_01231), were used as internal controls (Kim and Yun, 2011 ; Son et al., 2013 (link)). The experiment was conducted with 3 biological replicates. The obtained data were analyzed using Bio-Rad CFX Manager 3.0 software. Semi-quantitative RT-PCR was performed with a 20 μl reaction mixture containing 20 ng of cDNA, TaKaRa Taq™ (Takara Bio, Japan), and 10 pmoles of each primer set. The reaction condition was as follows: 3 min at 95 °C and 25 or 30 cycles of 25 s at 95 °C, 30 s at 57 °C, and 20 s at 72 °C, and the final extension was conducted at 72 °C for 7 min. Amplified PCR product was visualized on 1% agarose gel containing ethidium bromide under UV light after gel electrophoresis. All primer sets are listed in Table S2.
3
Nested PCR Amplification of β-Globin Gene
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Genomic DNA was extracted from axillary lymph nodes using SepaGene (EIDIA, Tokyo, Japan) according to the protocol recommended by the manufacturer. Two sets of primers for the human β-globin gene were designed (human β-globin Primer Set; Takara Bio, Shiga, Japan). The first primers were designed outside the region amplified by the second primer. The outer primers were designed as follows: GH20 (forward) (5′-GAA GAG CCA AGG ACA GGT AC-3′ ) and GH21 (reverse) (5′-GGA AAA TAG ACC AAT AGG CAG-3′ ) (amplified size of DNA: 408 bp); and the inner primers, KM29 (forward) (5′-GGT TGG CCA ATC TAC TCC CAG G-3′ ) and KM38 (reverse) (5′-TGG TCT CCT TAA ACC TGT CTT G-3′ ) (262 bp). The PCR reaction contents were as previously described using TaKaRa Taq (Takara Bio). The PCR conditions for the first reaction were 30 cycles of denaturing at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 2 min, according to the protocol recommended by the manufacturer. In the second step, the first PCR product was removed and reamplified using two sets of overlapping inner primers. Conditions for the second PCR were the same as for the first reaction. The PCR product was electrophoresed in a 2.0% agarose gel containing 0.5 µg/mL ethidium bromide [19] (link)–[22] (link).
4
Quantification of Cathelicidin Expression
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The reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to determine the expression of CATHs in the proventriculus and cecum using the cDNA of the chicks from control group of Experiment 1 (0-LPS of non-P-group; n=5). The reaction mixture (25 µL) containing 0.5 µL of cDNA, 1×PCR buffer, 0.2 µMdNTP mixture, 0.5 µM of each primer (forward and reverse), and 0.125U Takara Taq (Takara Bio. Inc., Shiga, Japan) was prepared. CATHs primers used for PCR are presented in Table 1 . The PCR cycle parameters were 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for all CATHs for 45 s, and extension at 72°C for 45 s, followed by a final extension at 72°C for 7 min. The PCR products were separated by electrophoresis on 2% (wt/vol) agarose gels containing 0.025% (wt/vol) ethidium bromide and photographed under UV illumination.
5
Quantification of Stem Cell and Lineage Markers
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Reverse transcription polymerase chain reaction (RT-PCR) and quantitative PCR were used to determine the mRNA expressions of stem cell (oct4, sox2, p75ntr) and osteogenic (runx2, ocn) / tenogenic (scleraxis, aggrecan) / chondorgenic (sox9, decorin) / adipogenic (pparγ, lpl) differentiation markers. Total RNA was extracted with Trizol-reagent (Invitrogen), First-strand complementary DNA synthesis was performed by SuperScript First-Strand Synthesis (Invitrogen). Complementary DNA was amplified with Takara-Taq (Takara Bio Inc.). PCR reactions were performed with Mx3000P QPCR System (Agilent Technologies). Human-specific primer sets are shown in Table 1 . As internal standards, glyceraldehyde-3-phosphate dehydrogenase (gapdh) and hypoxanthine phosphoribosyl transferase 1 (hprt1) primers were used for RT-PCR and quantitative PCR, respectively.
6
Genomic DNA Amplification and Sequencing
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Genomic DNA was prepared from the whole body or tail tip using Quick-gDNA Miniprep (Zymo Research). Up to 500 ng of genomic DNA was used as template for PCR to amplify the region surrounding the TRα TALEN target site (Takara Taq; Takara Bio Inc) that contained 0.2 μM of forward (5′-GGTTTCTTYCGCCGCACCA) (Y stands for C or T) and reverse (5′-ATCCATTGCCATGCCAACGG) primers with the following reaction conditions: 94°C for 5 minutes, 33 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. PCR products were spin column purified (Qiagen), cloned into TOPO T vectors (Invitrogen), and sequenced.
7
Quantifying Rspo3 Expression in Muscle
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Total RNA was extracted from each muscle fibre type and tissues (SOL, TA, EDL, ovary, lung) using Trizol reagent (Invitrogen, CA, USA). The presence of Rspo3 transcript was analysed by conventional PCR (RT-PCR). PCR was performed using TaKaRa Taq (TaKaRa, Shiga, Japan) with specific primer pairs. Primer sequences for this study can be found in Supplementary Table 1 . Quantitative real-time PCR (qRT-PCR) was performed on a 96-well PikoReal Real-Time PCR System with a DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. The mRNA levels of each gene were normalised to those of the housekeeping gene GAPDH and TATA binding protein (Tbp). Primer sequences for this study can be found in Supplementary Table 2 . The mRNA content of both Rspo3 and Tbp was calculated from the cycle threshold values using a standard curve, and the ratio between Rspo3 and Tbp was calculated. Primers were synthesised by Eurofins Genomics Co., Ltd. (Tokyo, Japan).
8
Quantifying DNA Methylation by COBRA
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Genomic DNA was extracted from the whole bodies of 18.5 dpc embryos and the livers of 5-week-old mice by phenol/chloroform extraction. Purified DNA samples (500 ng) were processed using an Epitect Plus DNA Bisulfite Kit (QIAGEN) according to the manufacturer’s instructions. Modified DNA was amplified using TaKaRa Taq (TaKaRa, Kusatsu, Japan) and the PCR primers described in Additional files 2 and 3 . Percentages of demethylated CpG sites were determined by COBRA. Briefly, amplified fragments were cleaved with restriction enzymes (Additional files 2 and 3 ) whose recognition sites were located at the CpG sites, and then separated and quantified using capillary and microchip electrophoresis (MCE-202 MultiNA, Shimadzu, Kyoto, Japan). Methylation levels were calculated as the percentage of cleaved DNA (mV･μm) among total DNA (mV･μm).
9
Multiplex DPO PCR for Influenza Strain Detection
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Multiplex DPO PCR reactions contained 1.5 U of Takara Taq (Takara Bio, Dalian, China), 2.5 μl of 10× PCR buffer, 2.0 mM Mg2+ and 250 μM dNTPs. The optimal concentration of DPO primer mix are listed in Table 2 . 2009 pandemic H1N1, seasonal H1N1, H3N2 and H5N1 primers at a concentration of 0.8 μM and influenza B primers at a concentration of 1.6 μM, 2 μl cDNA and nuclease-free water were added to a total volume of 25 μl. The amplification conditions of the multiplex PCR were as follows: pre-denaturation step for 5 min at 94°C, 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, followed by a final extension step at 72°C for 10 min. The multiplex DPO PCR was performed with an Applied Biosystems® 2720 Thermal Cycler (Life Technologies, NY, USA). The amplified products were analyzed by 2.0% agarose gel electrophoresis.
| Forward Primer (μM) | 20 | 20 | 20 | 20 | 40 |
| Reverse Primer (μM) | 20 | 20 | 20 | 20 | 40 |
| 25× primer mix (μM) | 0.8 | 0.8 | 0.8 | 0.8 | 1.6 |
10
Oak Tree Microsatellite Marker Amplification
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All PCR reactions were carried out in an Eppendorf thermal cycler with a 10 μl reaction mixture composed of 2 μl of DNA (10 ng/μl), 4 pmol of each forward and reverse primers, 25 mM MgCl2, 10 mM dNTP, 1 μl of 10× Mg free PCR reaction buffer, 1 μl of 4% BSA, 0.25 U/μl TaKaRa Taq™ (Takara Bio USA, Mountain View, California) and 3.5 μl of double distilled H2O. The PCR amplification profile consisted of initial denaturation at 94 °C for 2 min, 35 cycles of 94 °C for 30 s, annealing at a marker specific temperature for 30 s, then 72 °C for 45 s followed by 60 °C for 45 min and ending with a final extension at 72 °C for 10 min. Fluorescently labeled amplicons were size fractionated on an ABI 3730 XL genetic analyzer (Applied Biosystems, Foster City, CA) using GeneScan™ 400 HD ROX™ (Applied Biosystems) as internal size standard. Fragment length polymorphisms were scored using GeneMapper® v 4.0 (Applied Biosystems). Of the 379 SSR markers tested (67 gSSRs from Q. rubra, 180 EST-SSRs from Q. rubra, 120 bin-mapped EST-SSRs from Q. robur and 12 EST-SSRs from C. mollissima), 116 markers were informative (Additional file 1 ).
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