RNA was prepared from confluent tilapia lip OmL cultures using Trizol (ThermoFisher). cDNA was synthesized from it using a New England BioLabs (Beverly, MA) AMV First Strand cDNA Synthesis kit and, with primers given in S2 Table , used as a template for PCR amplification of the full length coding regions of Tgm1A and Tgm1B. The PCR products were cloned into pCDNA3.1 using a Gibson Assembly kit from New England BioLabs. Constructs were verified by sequencing of the 5’ and 3’ ends. A 3X FLAG epitope, encoding the peptide sequence DYKDHDGDYKDHDDYKDDDDK , was added at the 3’ end of the Tgm1 sequences by cloning double stranded oligonucleotides with appropriate overhangs into the XhoI and Asp718 sites of the cDNA constructs. Constructs with deletion of the 5 amino acids in the cysteine cluster (CPCCC) or their replacement by AIAAA (with an MscI restriction site introduced for screening) were prepared using the New England BioLabs Q5 Site-Directed Mutagenesis Kit (primers given in S3 Table ). HEK293FT cells (Invitrogen) were transfected with the cDNAs using Lipofectamine 2000 (Thermo-Fisher).
Amv first strand cdna synthesis kit
Manufactured by New England Biolabs
Sourced in United Kingdom, United States
The AMV First Strand cDNA Synthesis Kit is a laboratory product that enables the conversion of RNA into complementary DNA (cDNA). It utilizes the Avian Myeloblastosis Virus (AMV) reverse transcriptase enzyme to perform this conversion process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (4 mentions)
Trizol plus rna purification system, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by New England Biolabs (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Jumpstart sybr green supermix, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Gibson assembly kit, by New England Biolabs (1 mentions)
Hek293ft cells, by Thermo Fisher Scientific (1 mentions)
Expand high fidelity pcr system, by Roche (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Unlq 10 column trizol total rna extraction kit, by Sangon (1 mentions)
Sequence detection software version 1, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 7300 detection system, by Thermo Fisher Scientific (1 mentions)
Hp total rna isolation kit, by Omega Bio-Tek (1 mentions)
20 protocols using amv first strand cdna synthesis kit
1
Cloning and Characterization of Tilapia Tgm1 Proteins
2
Quantification of SIAH gene family
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Total RNA was isolated from cancer cell lines using RNeasy Mini Kit per the manufacturer’s instructions and extraction protocol (Qiagen. Germantown, MD). cDNA synthesis was carried out using AMV First Strand cDNA synthesis kit following the manufacturer’s protocol (New England BioLabs. Ipswich, MA). PCR amplification was performed using Expand High Fidelity PCR System (Roche. Indianapolis, IN). All primers were purchased from Integrated DNA Technologies (Coralville, IA).
The forward and reverse primers for PCR amplification of the siah1 cDNA transcript were 5′-ATGAGCCGTCAGACTGCTACAG-3′ and 5′-CAGGACTGCATCATCACCCAGT-3′, respectively. The forward and reverse primers for PCR amplification of the siah2 cDNA transcript were 5′-GCCATCGTCCTGCTCATTGGCA-3′ and 5′-ACCAATATGGGAAGGCAGGCAGGAAGGGGC-3′, respectively. The forward and reverse primers for PCR amplification of the siah3 cDNA transcript were 5′-ATGCTTTTCTTTACCCAGTGCT-3′ and 5′-TCACATTTCAGCTTCTGAGGGGA-3′, respectively. The forward and reverse primers for PCR amplification of the gapdh cDNA transcript were 5′-AAAGGGTCATCATCTCTGCC-3′ and 5′-TGACAAAGTGGTCGTTGAGG-3′, respectively.
The forward and reverse primers for PCR amplification of the siah1 cDNA transcript were 5′-ATGAGCCGTCAGACTGCTACAG-3′ and 5′-CAGGACTGCATCATCACCCAGT-3′, respectively. The forward and reverse primers for PCR amplification of the siah2 cDNA transcript were 5′-GCCATCGTCCTGCTCATTGGCA-3′ and 5′-ACCAATATGGGAAGGCAGGCAGGAAGGGGC-3′, respectively. The forward and reverse primers for PCR amplification of the siah3 cDNA transcript were 5′-ATGCTTTTCTTTACCCAGTGCT-3′ and 5′-TCACATTTCAGCTTCTGAGGGGA-3′, respectively. The forward and reverse primers for PCR amplification of the gapdh cDNA transcript were 5′-AAAGGGTCATCATCTCTGCC-3′ and 5′-TGACAAAGTGGTCGTTGAGG-3′, respectively.
3
RNA Extraction and RT-qPCR Quantification
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RNA was isolated as described above. RNA was reverse transcribed using the AMV First‐Strand cDNA Synthesis Kit (NEB Inc., Ipswitch, MA) according to manufacturer's instructions. RT‐qPCR was performed using a SensiFAST SYBR No ROX Kit (Bioline USA Inc., Taunton, MA, USA). Primers were designed using Primer3 software (Koressaar and Remm, 2007 ; Untergasser et al., 2012 ) for the amplification of gene fragments of approximately 100–200 bp in length and with an annealing temperature of 60°C (Table S7). RT‐qPCR was performed on a CFX96 real‐time PCR system (Bio‐Rad, Hercules, CA). The run conditions were 2 min of initial denaturation at 95°C, 95°C for 5 s, 58°C for 10 s and 72°C for 20 s (40 cycles). The relative expression of genes was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001 ) with the soybean gene GmCon15S (Libault et al., 2008 ) as endogenous control. Three biological replicates were used for each sample.
4
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA from tissue samples and from cell-line pellets was extracted using TRIzol Plus RNA Purification System (Life Technologies, Paisley, UK), and quantified by NanoDrop ND-1000 (Thermo Fisher Scientific, Loughborough, UK). Total RNA was reverse transcribed using AMV First Strand cDNA synthesis kit (New England Bio Labs, Hertfordshire, UK) after DNase treatment (DNase I (#M0303), New England Bio Labs, Hertfordshire, UK), using the manufacturer’s protocol as previously described [34 (link)]. cDNA was amplified by qPCR using JumpStart SYBR Green supermix (Sigma, Dorset, UK) and CFX Connect Real-Time System (Bio-Rad, Hertfordshire, UK) and the following primers: AGR2 amplification were: forward 5′-ATTGGCAGAGCAGTTTGTCC-3′, reverse 5′-GAGCTGTATCTGCAGGTTCGT-3′ [45 (link)]; for Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ), forward 5′-CGTTACTTGGCTGAGGTTGCC-3′, reverse 5′-GTATGCTTGTTGTGACTGATCGAC-3′ [46 (link)]; and for Peptidylprolyl Isomerase A (PPIA), forward 5′-AGACAAGGTCCCAAAGAC-3′ and reverse 5′-ACCACCCTGACACATAAA-3′ [47 (link)]. Relative transcript expression was calculated using the ΔΔCT method, normalised to the reference genes YWHAZ and PPIA using Biorad CFX manager.
5
Quantitative Analysis of NHE1 mRNA Expression
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RT-qPCR was used to measure the NHE1 mRNA expression levels. Briefly, the hearts were harvested following reperfusion, total RNA was extracted from the left ventricular heart tissues using TRIzol reagent and the RNA purity was quantified spectrophotometrically at a ratio of 260 to 280 nm. cDNA was obtained from 1 µg total RNA using the AMV First Strand cDNA Synthesis kit according to the manufacturer's instructions (New England Biolabs Inc., Ipswich, MA, USA). Quantitative (real-time) PCR (qPCR) was performed using iQ SYBR-Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The NHE1 and GAPDH (internal control) primer sequences were defined as follows: NHE1 forward, 5′-GCGGCGAGCAGATCAATAA-3′ and reverse, 5′-ACAGTGACGGCATCGTTGAG-3′; and GAPDH forward, 5′-CAAGTTCAACGGCACAGTCAA-3′ and reverse, 5′-CGCCAGTAGACTCCACGACA-3′. The reaction conditions were as follows: 10 sec at 95°C, 40 sec at 60°C and 45 sec at 72°C for 40 cycles. Amplification of the products was followed by melting curve analysis using Applied Biosystems 7500 system software, as previously described (28 (link)) (Life Technologies, Grand Island, NY, USA). The value of NHE1 mRNA was expressed relative to that of GAPDH from the same sample.
6
Transcriptional Analysis of T. versicolor Mycelia
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On day 18 of liquid fermentation, the mycelia of T. versicolor in the co-culture and mono-culture, were harvested and stored at −80 °C, respectively. The mycelia were ground to powder under liquid N2. Total RNA was extracted according to standard protocols of the UNlQ-10 column Trizol Total RNA Extraction Kit (Sangon, China) and cDNAs was obtained using the AMV First Strand cDNA Synthesis Kit (NEB, USA). The single stranded cDNA was 1/10 diluted for qPCR analysis using the primer pairs (Supplementary Table S5 ) and 2X SG Fast qPCR Master Mix (High Rox) (Sangon, China) for each of the six candidate genes. GAPDH was employed as a reference gene49 (link). qPCR was performed in accordance with the manufacturer’s instructions. Each reaction was conducted in biological triplicate. The Ct values obtained were used as the original data to calculate the relative transcriptional expression level of the candidate genes normalized against that of GAPDH gene. In correlation analysis, we normalized the genes expression levels by the 2−△△Ct method50 (link). The qRT-PCR product was also identified by 2% agarose gel electrophoresis.
7
Quantitative Real-Time PCR for Gene Expression
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Total RNA from tissue samples was extracted using TRIzol Plus RNA Purification System (Life Technologies, Paisley, UK), and quantified by NanoDrop ND-1000 (Thermo Fisher Scientific, Loughborough, UK). Total RNA was reverse transcribed using AMV First Strand cDNA synthesis kit (New England Bio Labs, Hertfordshire, UK) after DNase treatment (DNase I (#M0303), New England Bio Labs, Hertfordshire, UK), using the manufacturer's protocol. cDNA was amplified by qPCR using JumpStart SYBR Green supermix (Sigma, Dorset, UK) and the Light Cycler 96 Roche Real-Time System (Roche Diagnostics Ltd. BurgessHill, UK). Primers are listed in Supplementary Table 2 . Relative transcript expression was calculated by the ΔΔCT method, normalised to the reference gene YWHAZ (Sadek et al, 2011 (link)) using Biogazelle qbase+ software (Biogazelle NV, Zwijnaarde, Belgium)
8
Quantitative Real-Time PCR Gene Expression Analysis
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Total RNA was isolated using Trizol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Reverse transcription reactions were performed using the AMV First Strand cDNA Synthesis Kit (NEB, USA). Real-time PCR assays were performed using an Applied Biosystems 7300 Detection System (Applied Biosystems®, CA). The real-time PCR reaction was performed according to the protocol of the Power SYBR Green PCR Master Mix (Applied Biosystems, CA). The amplification procedure: 95°C for 15 seconds, and 60°C for 60 seconds for 40 cycles. Data was analyzed using the Sequence Detection Software, Version 1.2.3 (Applied Biosystems). The amount of target cDNA was calculated and normalized with the GAPDH levels in the samples. The primers utilized for real-time PCR analysis were listed in Table S2 .
9
RNA Extraction and cDNA Synthesis for Onchocerca
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Whole RNA of O. ochengi male, female, mff (skin and uterine) and L3 were extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) including the column DNAse digest. First strand cDNA synthesis was executed with the AMV First Strand cDNA Synthesis kit (New England Biolabs) using 500 ng RNA and random-hexamer primers according to the manufacturer’s instruction.
10
Extraction and Analysis of CACNA1C Transcripts
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Human lung tissue (8 donors) without overt disease, but not suitable for transplant, was obtained from the Life Alliance Organ Recovery Agency according to institutional review board guidelines regarding consent and de-identification of individual donors (demographic characteristics, see Table 1 ). Peripheral lung tissue was excised, chopped into ~ 5 mm fragments, and snap frozen in liquid N2. For preparation of total RNA, tissue was ground in a mortar and pestle under liquid N2 and the powder immediately extracted using E.Z.N.A. HP Total RNA Isolation Kit (Omega Bio-Tek, Norcross, GA). Total RNA was treated with DNase I and reverse transcribed using AMV First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA) with a primer specific in exon 50 of the CACNA1C transcript 3′ UTR (see Table 1 for primers sequences). cDNA was amplified by 35 cycles of PCR using Hotstar Hifidelity Taq polymerase kit (Qiagen, Valencia, CA) with primers specific for exons surrounding known spice sites (Table 2 ) and the PCR reactions cloned into pGEMTeasy. cDNA in random individual colonies was isolated and sequenced.
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