Premier 5

Manufactured by Premier Biosoft

Sourced in United States

Premier 5.0 is a software application developed by Premier Biosoft for analyzing and interpreting biological data. The software provides tools for tasks such as DNA and protein sequence analysis, primer design, and phylogenetic tree generation.

Automatically generated - may contain errors

Lab products found in correlation

Rnaiso plus, by Takara Bio (3 mentions) Sybr premix ex taq 2, by Takara Bio (3 mentions) Primescript rt reagent kit with gdna eraser, by Takara Bio (3 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Quantitative real time pcr, by Bio-Rad (2 mentions) Sybr premix ex taqtm 2 kit, by Takara Bio (2 mentions) Prim script rt reagent kit, by Takara Bio (2 mentions) Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions) Primescript rt reagent kit, by Takara Bio (2 mentions) Trizol reagent, by Takara Bio (2 mentions) Sybr premix ex taq, by Takara Bio (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Qtower 3 real time pcr thermal cycler, by Analytik Jena (1 mentions) Rq1 dnase, by Thermo Fisher Scientific (1 mentions) Rneasy plant mini kit, by Qiagen (1 mentions) Lightcycler 480 2 real time pcr system, by Roche (1 mentions) Goscript reverse transcription system, by Promega (1 mentions) Abi quantstudio 6 flex, by Thermo Fisher Scientific (1 mentions) Rnaiso plus reagent, by Takara Bio (1 mentions) Nanodrop nd 2000, by Thermo Fisher Scientific (1 mentions)

46 protocols using premier 5

Quantitative RT-PCR Analysis of Oxidative Stress

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Samples were rinsed by pre-frozen Diethypyrocarbonate (DEPC) water and then frozen in liquid nitrogen at −80 °C. Approximately 60 mg of frozen sample was ground thoroughly in a precooling tissue homogenizer. Total RNA was extracted by a commercial kit (TRIzol. Invitrogen, Shanghai, China). The RNA concentration was detected by a nucleic acid protein analyzer which had the D260/D280 reverse transcription eligible range at 1.8–2.0. The cDNA was stored at −80 °C.
According to the operator’s manual, QRT-PCR could be used to detect the expression of Nrf2, NQO1, HO-1, Bax and Bcl-2, and β-actin could be used for normalizing the expression of aforementioned genes as an endogenous control. In this study, the expression of the aforementioned genes in CG was defined as baseline (value = 1), and the 2−ΔΔCt method was applied for quantifying gene expressions, where ΔΔCt is the detected ΔCt- reference ΔCt and ΔCt is the Ct _{target gene} − Ct _{housekeeping gene}. Primers used in the present study (Supplementary Table S2) were designed by Premier 5 (PREMIER Biosoft International, New York, NY, USA) and synthesized by Chengke BioTech Co., Ltd. (Guangzhou, China).

Xu D., Yin L., Lin J., Fu H., Peng X., Chang L., Zheng Y., Zhao X, & Shu G. (2021). Aristolochic Acid I-Induced Hepatotoxicity in Tianfu Broilers Is Associated with Oxidative-Stress-Mediated Apoptosis and Mitochondrial Damage. Animals : an Open Access Journal from MDPI, 11(12), 3437.

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Validating RNA-Seq Results by qPCR

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Primers were designed using premier 5 (Premier Biosoft International, Palo Alto, CA, USA) and synthesized at BGI-Tech (Shenzhen, China). All the information for each primer pair can be found in the Additional file 3: Table S1. RNA-Seq results, including differential expression and NATs were validated by qPCR with SYBR® Premix Ex Taq ™ II (TaKaRa, Cat. # RR820A, Japan) on Applied Biosystems QuantStudio™ 6 Flex Real-Time PCR System (ThermoFisher, USA). For each experiment, NTB or UBQ was analyzed in triplicates as the positive control [95 (link)].

Zhang H., Wang H., Zhu Q., Gao Y., Wang H., Zhao L., Wang Y., Xi F., Wang W., Yang Y., Lin C, & Gu L. (2018). Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications. BMC Plant Biology, 18, 125.

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Ileum Mucosa Transcriptome Analysis

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The samples of ileum mucosa were washed with prefrozen diethyl pyrocarbonate water and immediately frozen in liquid nitrogen and stored at −80°C. Approximately 60 mg of the preserved sample was ground thoroughly with liquid nitrogen in a precooled mortar. Total RNA was extracted with TRIzol (Invitrogen). RNA concentration was determined by a nucleic acid protein analyzer, of which the D260/D280 range eligible for reverse transcription was 1.8–2.0. The cDNA was stored at −80°C.
According to the specific steps of SYBR Green Remix Ex Taq kit specification of TaKaRa and with GAPDH as the internal reference, QRT-PCR was used to detect the expression levels of the following genes: Claudin1, Zo1, Muc2, Nrf2, HO-1, Caspase 3, Caspase 9, Bax, Bcl-2, VDAC, TNF-α, and IFN-γ. The 2^−△△CT method was used to calculate the relative expression levels of these genes. All primers (Supplementary Table 2) were designed using Premier 5 (PREMIER Biosoft International, Palo Alto, CA) and synthesized by Chengke BioTech Co., Ltd.

Shu G., Xu D., Ran C., Yin L., Lin J., Fu H., Zhang W., Bai S., Peng X., Zhao X, & Amevor F.K. (2020). Protective effect of dietary supplementation of Bupleurum falcatum L saikosaponins on ammonia exposure–induced ileum injury in broilers. Poultry Science, 100(3), 100803.

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Liver RNA Expression Analysis

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The liver samples were stored at −80 °C for RNA extraction. The total liver RNA was extracted using RNAiso Plus (9108, Takara, Otsu, Japan) by following the manufacturer’s instructions. cDNA was synthesized simultaneously using 1 ug of RNA with the Prim-Script TM RT Reagent Kit (9108, Takara, Japan) and the SYBR Premix Ex Taq TM II Kit (9108A, Takara, Japan). The expression levels of Nrf2, HO-1, CAT, GSH-Px, and SOD-1 were detected by real-time quantitative PCR (Bio-Rad, Hercules, CA, USA). β-actin was applied as a housekeeping gene for normalization. The RNA expression levels were calculated using the 2^−ΔΔCT method [30 (link)]. The primers used in this study were designed by PREMIER 5 (PREMIER Biosoft International, Palo Alto, CA, USA) and are shown in Supplementary Data Table S1.

Zhang S., Zheng Y., Du H., Zhang W., Li H., Ou Y., Xu F., Lin J., Fu H., Ni X., Chang L.J, & Shu G. (2023). The Pathophysiological Changes and Clinical Effects of Tetramethylpyrazine in ICR Mice with Fluoride-Induced Hepatopathy. Molecules, 28(12), 4849.

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Kidney RNA Extraction and qRT-PCR Analysis

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The kidney samples were washed with pre-frozen DEPC water and immediately frozen in liquid nitrogen and stored at −80 °C. Approximately 60 mg of preserved sample was ground thoroughly with liquid nitrogen in a precooled mortar. Total RNA was extracted with TRIzol (Invitrogen, Shanghai, China). The RNA concentration was determined by a nucleic acid protein analyzer, of which the D260/D280 range eligible for reverse transcription was 1.8–2.0. The cDNA was stored at −80 °C.
According to the specific steps of the SYBR Green Remix Ex TaqTM kit specification of TaKaRa, QRT-PCR was used to detect the expression levels of the following genes: Claudin1, NQO1, HO-1, Caspase 3, Bax, Bcl-2 and Raf-1. β-actin was used as the endogenous control to normalize the expression of genes. We chose the CG group (blank group) as a reference group (value = 1), and the fold change of gene expressions was quantified using the 2^−ΔΔCt method, where ΔCt = Ct _{target gene} − Ct _{housekeeping gene}, and ΔΔCt = ΔCt − ΔCt _reference. All primers (Supplementary Table S2) were designed using Premier 5 (PREMIER Biosoft International, New York, NY, USA) and synthesized by Chengke BioTech Co., Ltd, Guangzhou, China.

Xu D., Ran C., Yin L., Lin J., Fu H., Peng X., Zhao X, & Shu G. (2021). Acute and Subchronic Toxicity Studies of Aristolochic Acid A in Tianfu Broilers. Animals : an Open Access Journal from MDPI, 11(6), 1556.

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Liver Gene Expression Analysis

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Liver blood was washed with prefrozen DEPC water and immediately frozen in liquid nitrogen for storage. Approximately 60 mg preserved liver was ground thoroughly with liquid nitrogen in a precooled mortar. Total RNA was extracted with TRIzol. RNA concentration was determined by a nucleic acid protein analyzer, of which the D260/D280 range eligible for reverse transcription was 1.8–2.0. cDNA was stored at −80 °C.
According to the specific steps of SYBR Green Remix Ex Taq^TM kit specification of TaKaRa and with β-actin as the internal reference, QRT-PCR was used to detect the expression levels of the genes Gadd45a, Mapk7, and Rras2, and the 2^−ΔΔCT method was used to calculate the relative expression levels of these genes. All primers (Table 1) were designed using Premier 5 (PREMIER Biosoft International, USA) and synthesized by Chengke BioTech Co., Ltd.

Lin S.Y., Xu D., Du X.X., Ran C.L., Xu L., Ren S.J., Tang Z.T., Yin L.Z., He C.L., Yuan Z.X., Fu H.L., Zhao X.L, & Shu G. (2019). Protective Effects of Salidroside against Carbon Tetrachloride (CCl4)-Induced Liver Injury by Initiating Mitochondria to Resist Oxidative Stress in Mice. International Journal of Molecular Sciences, 20(13), 3187.

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Quantifying Hepatic Antioxidant Gene Expression

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The livers samples were stored at −80°C for RNA extraction. Hepatic total RNA was extracted by RNAiso Plus (9108, Takara, Otsu, Japan) following the manufacture's instructions. cDNA was synthesized using one microgram of RNA concurrently with the Prim-ScriptTM RT reagent Kit (9108, Takara, Japan) and SYBR Premix Ex TaqTM II kit (9108A, Takara, Japan). The RNA expressions, including Nrf2, HO-1, GST, GSH-Px, SOD1, CAT, and NQO-1, were quantified by Real-Time quantitative PCR (Bio-Rad, USA). β-actin was used as the house-keeping gene to normalize the gene expression, and the 2-^ΔΔCT method was used for calculation of the RNA expression levels (34 (link)). The primers (Table 1) were designed by using of Premier 5 (PREMIER Biosoft International, Palo Alto, CA) and synthesized by Qingke BioTech Co., Ltd (33 (link)).

Du H., Zheng Y., Zhang W., Tang H., Jing B., Li H., Xu F., Lin J., Fu H., Chang L, & Shu G. (2022). Nano-Selenium Alleviates Cadmium-Induced Acute Hepatic Toxicity by Decreasing Oxidative Stress and Activating the Nrf2 Pathway in Male Kunming Mice. Frontiers in Veterinary Science, 9, 942189.

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qRT-PCR Validation of RNA-seq Data

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A total of 12 DEGs associated with berry development were selected for qRT-PCR validation of RNA-seq gene expression data (Supplementary Table 1). The same RNA used in RNA-seq was reverse transcribed using MonScript RTIII Super Mix with dsDNase (Monad, Wuhan, China). qRT-PCR was performed for three biologic replicates and three technical replicates using MonAmp ChemoHS qPCR Mix (Monad) on a qTOWER 3 Real-Time PCR Thermal Cycler (Analytik Jena, Jena, Germany). The primers used in this study were selected from the literature or designed using Premier 5 (Premier Biosoft, Palo Alto, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize all data, and relative expression levels were calculated with the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)).

Peng Y., Gu X., Zhou Q., Huang J., Liu Z., Zhou Y, & Zheng Y. (2022). Molecular and physiologic mechanisms of advanced ripening by trunk girdling at early veraison of ‘Summer Black’ grape. Frontiers in Plant Science, 13, 1012741.

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Molecular Detection of Tick-Borne Pathogens

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Primers were designed against regions of the A. bovis groEL gene and the A. phagocytophilum 16S rRNA gene using Premier 5 (Premier Biosoft International, Palo Alto, CA, United States). These regions were identified through alignments of nucleotide sequences obtained in this study and sequences available from the GenBank database (KX987399, KU585932, and KY425449 for A. bovis; KY242452, KR002115, LC060987, KF569915, and KC916737 for A. phagocytophilum). The specificity of each primer set was evaluated using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI) database¹. For the detection of A. capra and A. ovis targeting the groEL and msp4 genes, respectively, previously described primers were used (Torina et al., 2012 (link); Yang et al., 2016 (link)). The primers are described in Table 1. The primer sets were synthesized by Sangon Biotech Company (Shanghai, China).

Peng Y., Zhao S., Wang K., Song J., Yan Y., Zhou Y., Shi K., Jian F., Wang R., Zhang L, & Ning C. (2020). A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples. Frontiers in Microbiology, 11, 606.

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Identifying Markers for BoHL1 and BoHL2 in Cabbage

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First, we attempted to identify user-friendly markers from the fine mapping markers of BoHL1 and BoHL2 in the eight cabbage lines. We identified one SSR marker and 28 InDel markers for BoHL1 and 27 InDel markers for BoHL2 that have been used for the fine mapping of HL genes [19, (link)32] . Furthermore, all eight cabbage lines were resequenced to an ~20× depth to obtain additional nucleotide variations, which were deposited in the NCBI Sequence Read Archive (SRA) under BioSample accessions (SAMN06841129-30, SAMN17385836-47). For BoHL1, we searched the genomic region in the BoHL1 fine mapping region for nucleotide variations (SNPs, InDels and structural variants (SVs)) between the BoHL1 lines and no-BoHL1 lines (BoHL2 lines and three control lines); using the same strategy, we searched nucleotide variations between the BoHL2 lines and no-BoHL2 lines (BoHL1 lines and three control lines) from the fine mapping region of BoHL2. InDels ≥3 bp long and SVs were used to design InDel markers; InDels ≤2 bp long and SNPs were selected to design KASP markers following the PolyMarker pipeline). The primers were designed with Premier 5 (Premier Biosoft International, Palo Alto, CA, USA). Nucleotide variations associated with the markers were verified by Sanger sequencing, and sequence alignment was conducted with DNAman.

Xiao Z., Kong C., Han F., Yang L., Zhuang M., Zhang Y., Wang Y., Ji J., Li Z., Fang Z., & Lv H. (2021). Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea. Agronomy, 11(5), 982-982.

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