Primer premier software

Manufactured by Premier Biosoft

Sourced in United States, China

Primer Premier software is a computational tool for the design of oligonucleotides, such as primers and probes, for use in various molecular biology applications. The software provides algorithms for analyzing sequence data and generating optimal primer sequences based on user-specified criteria.

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Primer Premier Software v.5.0 Primer Premier software, version 3.0 Primer Premier Software 5.0 Primer Premier software version 5.0 Primer Premier Software version 6.0 Primer Premier

46 protocols using primer premier software

RNA Extraction and qPCR Analysis in Chicken

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Total RNA was isolated [2 (link)] from muscles, myoblasts, and embryos using TRIzol reagent according to the manufacturer's instructions (Invitrogen, Shanghai, China). The RNA preparation, qPCR, and relative mRNA abundance quantification procedures were the same as previously described [16 ]. The amplification efficiency for each gene was determined using the DART-PCR program [42 (link)]. The relative abundance of mRNA was also calculated [43 (link)] to account for gene-specific efficiencies and was normalized to the mean expression of GAPDH and β-actin.
Primer Premier Software (Premier Biosoft International, USA) was used to design specific primers based on known chicken sequences (Table 2).

Yao H., Fan R., Zhao X., Zhao W., Liu W., Yang J., Sattar H., Zhao J., Zhang Z, & Xu S. (2016). Selenoprotein W redox-regulated Ca2+ channels correlate with selenium deficiency-induced muscles Ca2+ leak. Oncotarget, 7(36), 57618-57632.

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Sequencing Accuracy of EsFem-1c 3'UTR

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To determine the sequence accuracy of EsFem-1c 3′UTR for future studies, the polymerase chain reaction (PCR) template and specific primers were utilized to perform the PCR (Table 1). Primer Premier software (version 6.0, Premier Biosoft, San Francisco, CA, USA) was used to design the primers [36 (link)].
A 25.0 µL mixture of approximately 0.5 µL of each primer (10 mM), 0.5 µL Ex Taq polymerase (TaKaRa, Japan), 1.0 µL cDNA template, 2.5 µL 10 × Ex Taq polymerase buffer, 2.0 µL dNTP, and 18.0 µL double-distilled water was used for the PCR. The PCR program included pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 80 s for 35 cycles. The products separated by gel electrophoresis were sequenced by Youkang (Yongkang, China). The sequences were trimmed of vector contamination using the VecScreen tool (https://www.ncbi.nlm.nih.gov/tools/vecscreen/ (accessed on 1 April 2023)) [37 (link)].
Poly (A) signal Miner (http://dnafsminer.bic.nus.edu.sg/PolyA.html (accessed on 1 April 2023)) was utilized to detect the position of the poly (A) signal [38 (link)]. The elements in 3′UTRs were analyzed using UTRScan (http://itbtools.ba.itb.cnr.it/utrscan (accessed on 1 April 2023)) [39 (link)].

Zhu D., Feng T., Mo N., Han R., Lu W, & Cui Z. (2023). Eriocheir sinensis feminization-1c (Fem-1c) and Its Predicted miRNAs Involved in Sexual Development and Regulation. Animals : an Open Access Journal from MDPI, 13(11), 1813.

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Quantitative PCR for Gene Expression

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Total RNA was extracted with TRIzol (Invitrogen) following the manufacturer’s instructions. RNA concentrations were determined with a Nano instrument (NanoDrop Technologies, Wilmington, DE). The first-strand cDNA was synthesized for each RNA sample using the Superscript III Reverse Transcriptase system (Invitrogen). Real-time quantitative PCR was performed on the iCycler (Biorad, UK) using the Quanti Tect SYBR Green PCR kit (Applied Biosystems). The forward and reverse primers for β-actin were designed using the Primer Premier software (Premier Biosoft International). The sequences of the PCR primer pairs were as follows: β-actin forward, 5′-GGA TGC AGA AGG AGA TCA CTG-3′ and reverse, 5′-CGA TCC ACA CGG AGT ACT TG-3′; TLR4 forward, 5′-AGT TTC CTG CAA TGG ATC AAGG-3′ and reverse 5′-CTG CTT ATC TGA AGG TGT TGC AC-3′; IL-6 forward, 5′-AGT GAG GAA CAA GCC AGA GC-3′ and reverse, 5′-CAG GGG TGG TTA TTG CAT CT-3′; IL-8 forward, 5′-GAC ATA CTC CAA ACC TTT CCA CCC-3′ and reverse, 5′-CCA GAC AGA GCT CTC TTC CAT CAG-3′. The human β-actin gene was used as an endogenous control for sample normalization. For each sample, the relative abundance of target mRNA was calculated from the obtained C_Δt values for both the target and endogenous reference β-actin genes using the 2^-ΔΔCt cycle threshold method.

Zhu Y., Dai B., Li Y, & Peng H. (2015). C5a and toll-like receptor 4 crosstalk in retinal pigment epithelial cells. Molecular Vision, 21, 1122-1129.

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Quantifying Gene Expression by qRT-PCR

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Total RNA was isolated by TRIzol (Invitrogen, Carlsbad, California, USA). The total RNA was treated with DNase I and reverse‐transcribed using oligo‐dT primers. The total cDNA was used as the starting material for real‐time PCR with FastStart Universal SYBR Green Master (Roche Applied Science, Mannheim, Germany) Germany) using the StepOne real‐time PCR System (Life Technologies Corp. Waltham, MA USA). The Primer Premier software (PREMIER Biosoft International, Palo Alto, California, USA) was used to design specific primers for TNF‐α, IL‐1β and IL‐6 and GAPDH based on known sequences (Table 1). The expression levels of each target gene were normalized to the corresponding GAPDH threshold cycle (CT) values using the 2^−▵▵CT comparative method.

Zhao G., Jiang K., Wu H., Qiu C., Deng G, & Peng X. (2017). Polydatin reduces Staphylococcus aureus lipoteichoic acid‐induced injury by attenuating reactive oxygen species generation and TLR2‐NFκB signalling. Journal of Cellular and Molecular Medicine, 21(11), 2796-2808.

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Quantitative Real-Time PCR for Gene Expression

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Total RNA was isolated from the tissue and PMN cell samples using the TRIzol reagent and synthesized first-strand cDNA using oligo(dT) primers and Superscript II reverse transcriptase, according to the manufacturer's instructions (Invitrogen, USA). Synthesized cDNA was diluted five times with sterile water and stored at -80°C. The Primer Premier software (PREMIER Biosoft International, USA) was used to design specific primers for LYSO and β-actin based on known sequences (Table 2). Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, USA). Reactions were performed in a 25-μl reaction mixture, under the following conditions: 95°C for 30 s, followed by 35 cycles of 95°C for 15 s, 63°C for 30 s and 60°C for 30 s. The results (fold changes) were expressed as 2^−ΔΔCt. β-actin served as the reference gene.

Gao X., Guo M., Zhang Z., Shen P., Yang Z, & Zhang N. (2017). Baicalin promotes the bacteriostatic activity of lysozyme on S. aureus in mammary glands and neutrophilic granulocytes in mice. Oncotarget, 8(12), 19894-19901.

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RNA Extraction and qPCR Analysis

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Total RNA was isolated from placenta samples and HTR-8/SVneo cells using TRIzol Reagent (Thermo Fisher Scientific, Inc.). Subsequently, RNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using a PrimeScript RT Master Mix kit (Takara Bio, Inc.), according to the manufacturer's protocol. The thermocycling conditions were as follows: 95°C for 10 min, 42°C for 2 min, 37°C for 15 min, 85°C for 5 sec and 4°C for 30 min. qPCR was performed using a SYBR PrimeScript RT-qPCR kit (Takara Bio, Inc.) on a Roche Light-cycler 480 real-time qPCR System (Roche Applied Science), according to the manufacturer's protocol. The thermocycling conditions of the qPCR were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. Primers for miR-320a, U6, IL-4 and GAPDH are presented in Table II. The primers were designed using Primer Premier software (version 5.0; Premier Biosoft International, Inc.), and purchased from Takara Biotechnology Co., Ltd. Expression level of miR-320a was normalized to the U6 expression level. IL-4 expression was normalized to GAPDH expression. Relative expression was calculated using the 2^−∆∆Cq method (21 (link)).

Xie N., Jia Z, & Li L. (2019). miR-320a upregulation contributes to the development of preeclampsia by inhibiting the growth and invasion of trophoblast cells by targeting interleukin 4. Molecular Medicine Reports, 20(4), 3256-3264.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA of the samples was extracted with an RNA extraction kit (Ai Weidi Biotechnology Co., Ltd., Shenzhen, China). cDNA was synthesized by Goldenstar RT6 cDNA Synthesis Kit Ver. 2 kit (Beijing TsingKe Biotech Co., Ltd., Beijing, China). qRT-PCR was performed using CFX96TM real-time system (Bio-Rad, CA, USA) with ChamQ SYBR qPCR Master Mix Kit (Vazyme Biotech Co., Nanjing, China) reagents. The amplification procedure was 95 °C pre-denaturation for 1 min; 95 °C denaturation 10 s, annealing at 58 °C for 15 s, and the number of amplification cycles was 39 cycles. Gene expression was normalized with AgACTIN as an internal control and calculated with 2⁻^ΔΔCt [49 (link)]. The primers for qRT-PCR were designed using Primer Premier software (version 6.0; Premier Biosoft International: Palo Alto, CA), and the primer sequences are listed in Table S5.

Li M., Li J., Zhang R., Lin Y., Xiong A., Tan G., Luo Y., Zhang Y., Chen Q., Wang Y., Zhang Y., Wang X, & Tang H. (2022). Combined Analysis of the Metabolome and Transcriptome to Explore Heat Stress Responses and Adaptation Mechanisms in Celery (Apium graveolens L.). International Journal of Molecular Sciences, 23(6), 3367.

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Transcriptome Analysis of Celery Under High Temperature

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The transcriptome data of celery under variable high temperature was obtained by this research group (Li et al., 2022a (link)), and the transcript abundance of AgHSF was calculated in log2(FPKM) to generated cluster heatmaps.
RT-qPCR was performed using SYBR Premix Ex Taq (TaKaRa, Dalian, China). All the steps were followed the manufacturer’s instruction (CFx384TM Real-Time System, Bio-Rad, USA), and the expression level was calculated by the 2^-△△Ct method (Pfaffla, 2001 (link)). AgTUB and AtACT2 were served as reference genes for celery and Arabidopsis genes, respectively. All gene primer sequences used in this study were designed using Primer Premier software (version 6.0; Premier Biosoft International: Palo Alto, CA), and the sequences are listed in Table S1.

Li M., Zhang R., Zhou J., Du J., Li X., Zhang Y., Chen Q., Wang Y., Lin Y., Zhang Y., He W., Wang X., Xiong A., Luo Y, & Tang H. (2023). Comprehensive analysis of HSF genes from celery (Apium graveolens L.) and functional characterization of AgHSFa6-1 in response to heat stress. Frontiers in Plant Science, 14, 1132307.

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Validating RNA Sequencing Data by RT-qPCR

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Quantitative real-time PCR was used to validate the reliability of RNA sequencing data as previously described [42 (link)]. The total RNA of the samples was extracted and reversed into cDNA for RT-qPCR analysis using applied biosystems 7500. The PCR procedure was pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, 40 cycles. Three biological replicates per reaction, relative expression levels calculated using the 2^−ΔΔCt method, with actin serving as the reference gene. Measurements were obtained in triplicate from three biological replicates. All primers were designed using Primer Premier software (version 5.0, PREMIER Biosoft, San Francisco, CA, USA) for the six unigenes (Supplementary Table S1).

Li Y., Gao Y., Deng L., Lian H., Guo W., Wu W., Xue B., Li B., Su Y, & Zhang H. (2023). Volatile Profiling and Transcriptome Sequencing Provide Insights into the Biosynthesis of α-Pinene and β-Pinene in Liquidambar formosana Hance Leaves. Genes, 14(1), 163.

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Integrin αvβ6 and IL-8 Expression Quantification

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Total RNA was isolated by TRIzol according to the manufacturer's protocol. Equal amounts of RNA were transcribed into cDNA using RNeasy plus micro kit. The total cDNA was used as starting material for real-time PCR with FastStart Universal SYBR Green Master (Roche Applied Science, Mannheim, Germany) on the StepOne real-time PCR System (Life Technologies Corp.). The Primer Premier software (PREMIER Biosoft International, USA) was used to design specific primers for integrin αvβ6, IL-8, and GAPDH based on known sequences. The primers for integrin αvβ6 were 5′-TTCCTAATGACGGGCTCTG-3′ (forward) and 5′-TTGGGTTACAGCGAAGATCA-3′ (reverse). The primers for IL-8 were 5′-CAATCCTAGTTTGATA CTCCC-3′ (forward) and 5′-AATTACTAATATTGACTGTGGAG-3′ (reverse). The expression levels of each target gene were normalized to corresponding GAPDH threshold cycle (CT) values using the 2^−ΔΔCT comparative method.

Yan P., Zhu H., Yin L., Wang L., Xie P., Ye J., Jiang X, & He X. (2018). Integrin αvβ6 Promotes Lung Cancer Proliferation and Metastasis through Upregulation of IL-8–Mediated MAPK/ERK Signaling. Translational Oncology, 11(3), 619-627.

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