Qualitative PCR oligonucleotide primers were designed using the software Primer Premier Version 5.00 (PREMIER Biosoft International, Palo Alto, CA, USA). qPCR primers and TaqMan fluorescent probes were designed using the software Beacon Designer 8.0 (PREMIER Biosoft, USA). All the primers and probes were synthesized by Sangon Biotech (Table S3 ).
Primer premier version 5
Manufactured by Premier Biosoft
Sourced in United States
Primer Premier version 5.0 is a software tool for designing and analyzing primers and probes for DNA and RNA amplification. The software provides a user-friendly interface for primer design, melting temperature calculation, and specificity analysis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
La taq enzyme, by Takara Bio (2 mentions)
Hiscript 3 all in one rt supermix perfect, by Vazyme (2 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
Beacon designer 8, by Premier Biosoft (1 mentions)
Hifair 3 1st strand cdna synthesis supermix for qpcr gdna digester plus, by Yeasen (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Hieff qpcr sybr green master mix no rox, by Yeasen (1 mentions)
Reverse transcriptase kit, by Takara Bio (1 mentions)
Hinc 2, by Takara Bio (1 mentions)
Mastercycler ep realplex detection system, by Eppendorf (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
M mlv platinum sybr green qpcr supermix udg kit, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Abi 3130 dna sequencer, by Thermo Fisher Scientific (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
32 protocols using primer premier version 5
1
Quantitative PCR Primer Design
2
Soybean Genome Sequence Analysis
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Soybean genome sequences were obtained from the Phytozome database (http://www.phytozome.net/ ). The Primer Premier Version 5.00 software package (Premier Biosoft International) was used to design primers and Multalin (http://multalin.toulouse.inra.fr/multalin/multalin.html ) for sequence alignments. An unrooted phylogenetic tree was constructed using MEGA 4.0 with the maximum likelihood (ML) method and bootstrap tests carried out with 1000 iterations [61 (link)]. The predicted sequence of pre-miR1511from other crops were confirmed for present of mature sequences and their secondary structure predicted using the RNAfold program (http://mfold.rna.albany.edu/?q = mfold). The Shannon diversity index was calculated as H = −sum (Pi ln[Pi]) where H represents the Shannon diversity index and Pi is the number of individuals in a particular species type divided by the total number of individuals of all types of species in the community.
3
Microsatellite Isolation and Characterization
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Microsatellites were isolated from a single specimen of R. venosa following an enrichment protocol described by Glenn and Schable [30] (link). Primers flanking the microsatellite repeat regions were designed using Primer Premier version 5.00 (PREMIER Biosoft International). Primers were optimized and checked for polymorphisms using a sample of 30 adults. The number of alleles (Na), observed (HO) and expected (HE) heterozygosity were calculated by using the Excel Microsatellite Toolkit [31] . Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) tests were performed with GENEPOP 4.0 [32] (link). Expected exclusion probabilities for each locus and across all loci were calculated with GERUD 2.0 [33] . The presence of null alleles for each locus was checked using Micro-Checker ver. 2.2.3 [34] . A total of five highly polymorphic loci were chosen for this study.
4
Quantitative Real-Time PCR Analysis of Algal Gene Expression
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Samples of algal cells grown under the photoheterotrophic and the autotrophic conditions were harvested and were used for qPCR validation. RNA extraction and RNA quality check were conducted as previously described. The Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus, Yeasen, Shanghai, China) was used for reverse transcription of 1 µg of total RNA according to manufacturers’ instructions. The quantitative real-time PCR (qRT-PCR) for gene expression assay was performed using Hieff® qPCR SYBR® Green Master Mix (No Rox) (Yeasen, Shanghai, China) on a quantitative thermal cycler (Light Cycler 480II, Roche, Basel. Switzerland). Specific primer pairs were designed for each target gene through Primer Premier (version 5.0) software (PREMIER Biosoft, San Francisco, CA, USA) using known sequences in the NCBI database (Supplemental Table S3 ). The qRT-PCR conditions were as follows: preincubation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, annealing temperature (corresponding specific primer pairs) for 20 s, and 72 °C for 20 s. Melting curves were systematically monitored (temperature gradient at 0.5 °C/s from 60 to 95 °C). Using 18S as reference genes, the genes were quantified using the 2−∆∆Ct value method according to Pfaffl (2001 (link)). The value of algal cell in the autotrophic condition was assigned as an arbitrary value of 1.
5
Gene Expression Quantification Protocol
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The primers specific for each target gene were designed based on exon distribution and mRNA sequence, using the Primer Premier version 5.0 software (Premier Biosoft, Palo Alto, CA, USA). Each primer spanned >two exons and yielded products of 100–250 bp in length. The primers and TaqMan® probes for IDO, HO-1, IL-7 and GAPDH were synthesized by Shenggong Biotech Co., Ltd. (Shanghai, China), and are presented in Table I . The housekeeping gene GAPDH was used as an internal reference.
6
Quantifying Renal KLF15 and NF-κB Expression
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The expression levels of KLF15 and NF-κB were determined in the renal tissue samples. The total RNA was extracted by using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), the concentration of total RNA was measured by microultraviolet spectrophotometer and 1.5 µg of total RNA was used for cDNA reverse transcription (at 37°C for 15 min) using a Reverse Transcriptase kit (D2639A; Takara Biotechnology Co., Ltd., Dalian, China). The total reaction mixture was 20 µl, made up of 10 µl SYBR Premix Ex Taq™ II X2 (cat. no. RR420A; Takara Biotechnology Co., Ltd.), 0.8 µl PCR forward primer (10 µM), 0.8 µl PCR reverse primer (10 µM), 0.4 µl ROX reference dye II X50, 6 µl UdH2O and 2 µl cDNA. The RT-qPCR cycling program was set at one cycle of pre-denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 34 sec, 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. The primer sequences were designed with the Primer Premier version 5.0 software (Premier Biosoft International, Palo Alto, CA, USA), and are presented in Table I . The relative levels of KLF15 and NF-κB mRNA were normalized to the β-actin levels, and calculated using the 2−ΔΔCq formula (18 (link)).
7
Genetic Polymorphism Analysis of Surfactant Proteins
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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyze SP-A, SP-B, and SP-D genetic polymorphisms. PCR primers were designed using Primer Premier version 5.0 software (Premier Biosoft, Palo Alto, CA) and synthesized by Shanghai Sangon Biological Engineering Technology Co., Ltd. (Shanghai, China). The primer sequences and their lengths are shown in Table 1 . PCR reaction was carried out in 20-μl volume, containing 10 μl×PCR PLUS MIX (DBI), 1.2 μl DNA templates, 0.5 μl upstream primer, and 0.5 μl downstream primer. PCR conditions were: 95°C initial denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 45 s, and 72°C extension for 50 s. After 35 cycles, the final extension was at 72°C for 10 min. PCR amplification products (6 ml) were digested with 4 U Fsp I (Toyobo) in a 15-l volume and incubated overnight at 37°C. A volume of 5 μl of the digested product was mixed with 3 μl 6×buffer solution and analyzed on 3% agarose gel, followed by UV photography to record the results. Locus-specific restriction enzymes HincII, XspI, and Ase I (TaKaRa) were also used to validate PCR products from different loci, and agarose gel electrophoresis was employed to analyze and record the results. All genotypes were finally confirmed by sequencing using a DNA sequencer (ABI370).
8
Gene Expression Quantification via qPCR
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Cells were homogenized in TRIZOL kit (Invitrogen, Carlsbad, CA) for extraction of RNA according to each manufacturer’s protocol. Both reverse transcription and quantitative PCR were carried out using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). Eppendorf Mastercycler ep realplex detection system (Eppendorf, Hamburg, Germany) was used for quantitative PCR analysis. The primers of genes including TNF-α, IL-6, IL-1β, VCAM-1, ICAM-1, and β-actin were synthesized by Invitrogen. PCR primers were designed using Primer Premier Version 5.0 software (Premier Biosoft, Palo Alto, CA, USA), and sequences were as follows (Invitrogen):
TNF-α sense: 5′-TGGAACTGGCAGAAGAGG-3′;
antisense: 5′-AGACAGAAGAGCGTGGTG-3′.
IL-6 sense: 5′-GAGGATACCACTCCCAACAGACC-3′;
antisense: 5′-AAGTGCATCATCGTTGTTCAT ACA-3′.
IL-1β sense: 5′-ACTCCTTAGTCCTCGGCCA-3′
antisense: 5′-CCATCAGAGGCAAGGAGGAA-3′
β-actin sense: 5′-TGGAATCCTGTGGCATCCATGAAAC-3′;
antisense: 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′.
VCAM-1 sense primer: 5′-TGCCGAGCTAAATTACACATTG-3′;
antisense primer: 5′-CCTTGTGGAGGGATGTACAGA-3′.
ICAM-1 sense primer: 5′-GCCTTGGTAGAGGTGACTGAG-3′;
antisense primer: 5′-GACCGGAGCTGAAAAGTTGTA-3′.
9
Primer Development for Environmental DNA
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TaqMan-based primer sets directed towards frogs were obtained from previously published information (eLICA1 [13 ] and eASMO [14 ]) or designed using organelle gene-specific sequence data from multiple species (NCBI Genome, http://www.ncbi.nlm.nih.gov/genome/organelle/ ). An analogous generation of primer sets was carried out on chloroplast gene sequences obtained from freshwater plant species. Table 1 identifies the mitochondrial and chloroplast gene sequences and their associated or confounding species used in primer development. For each gene target, species-specific sequences were aligned by ClustalW (http://www.genome.jp/tools/clustalw/ ) and the output aln file assessed using BioEdit (Ibis Biosciences, Carlsbad, CA, USA) and Primer Premier version 5 (Premier Biosoft, Palo Alto, CA, USA) for generation of either cross-species (ePlant5 and eFrog2, 3, and 5) or species-restricted (eLICA2 and eASMO9) primer sets. Note that the eLICA1 and eASMO hydrolysis probes were extended by four bases in the 3’ direction from that previously published [13 ,14 ] in order to allow for use of an increased annealing temperature to enhance stringency in the present eDNA assay (Table 2 ). DNA amplification primers and associated TaqMan hydrolysis probes were obtained from Integrated DNA Technologies (Coralville, IA, USA) and their characteristics are shown in Table 2 .
10
Transcriptional Analysis of Bacterial and Fish Immune Genes
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BaX030 was cultured in the two media for 20 h and centrifuged at 10000 rpm for 5 min to collect approximately 0.1 g of bacteria, and the total RNA of BaX030 was extracted by Trizol method (Yi et al., 2018 (link)). The RNA concentration was determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA), and the reverse transcription kit of the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used to perform genomic DNA removal and reverse transcription reactions. The relative expression level of each mRNA was normalized to 16S rRNA as the internal reference, The transcription levels of seven genes, leuA, zwf, leuD, secG, pdk2, pckA and gerPC, were detected.
To evaluate the mRNA expression level of fish liver and kidney, we selected the housekeeping gene β-actin as the reference gene (Wang et al., 2015 (link); Yang et al., 2016 (link)) and selected 5 immune-related genes, immunoglobulin M (IgM), complement C3 (C3), lysozyme (LSZ), interleukin-1β (IL-1β), and interleukin 8 (IL8)(Supplementary Table S1
To evaluate the mRNA expression level of fish liver and kidney, we selected the housekeeping gene β-actin as the reference gene (Wang et al., 2015 (link); Yang et al., 2016 (link)) and selected 5 immune-related genes, immunoglobulin M (IgM), complement C3 (C3), lysozyme (LSZ), interleukin-1β (IL-1β), and interleukin 8 (IL8)(
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