Multina

Manufactured by Shimadzu

Sourced in Japan, United States

The MultiNA is a laboratory instrument designed for the analysis and quantification of nucleic acids. It utilizes a microchip-based electrophoresis system to separate and detect DNA and RNA samples. The MultiNA provides rapid and high-resolution analysis of various types of nucleic acids, including PCR products, restriction fragments, and RNA.

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Rneasy mini kit, by Qiagen (3 mentions) Ez dna methylation gold kit, by Zymo Research (3 mentions) Primestar gxl dna polymerase, by Takara Bio (2 mentions) M mlv reverse transcriptase, by Promega (2 mentions) Random primer, by Takara Bio (2 mentions) Multiplex pcr kit, by Qiagen (2 mentions) Qubit fluorometer, by Thermo Fisher Scientific (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Zrob20 rna, by Next Advance (2 mentions) Tissuelyser 2 solution, by Qiagen (2 mentions) Ampure xp, by Beckman Coulter (2 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) E gel, by Thermo Fisher Scientific (2 mentions) Hpych4iv, by New England Biolabs (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Access rt pcr introductory system, by Promega (2 mentions) Dneasy blood tissue kit, by Qiagen (2 mentions) Biotaq hs dna polymerase, by Meridian Bioscience (2 mentions) Uvp blak ray xx 15l uv bench lamp, by Analytik Jena (2 mentions) Nebnext ultra 2 q5, by New England Biolabs (2 mentions)

Spelling variants of this product

MCE-202 MultiNA (49 citations) MCE-202 MultiNA Microchip Electrophoresis System (5 citations) MultiNA MCE-202 bioanalyzer (4 citations) MultiNA electrophoresis device (4 citations) MCE-202 MultiNA system (3 citations) MCE®-202 MultiNA device (3 citations) MultiNA capillary electrophoresis system (2 citations) Microchip Electrophoresis system for DNA/RNA Analysis MCE® -202 Multina (2 citations) MultiNA DNA-12000 kit (2 citations) MultiNA electrophoresis instrument (2 citations)

51 protocols using multina

Genomic DNA Extraction and NGS Analysis

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Genomic DNA was extracted using a DNeasy Blood and Tissue kit from the knock-in cells collected at 72 h post transfection. Preparation of libraries and NGS were performed by FASMAC. Briefly, NGS libraries were prepared via two-step PCR using the primers listed in Supplementary Data 1. The PCR amplification was performed using PrimeSTAR GXL DNA Polymerase (TakaRa) and the PCR products were purified using Agencourt AMPure XP (Beckman Coulter). Note that the PCR products amplified using PrimeSTAR Max DNA Polymerase were used as templates in the first-step PCR of PARP1 samples instead of genomic DNA because genomic PCR amplification failed in the PARP1 samples. NGS was performed using a MiSeq (Illumina). The PCR products were also analyzed by microchip electrophoresis using MultiNA ™ (hereinafter referred to as “MultiNA”) (Shimadzu) with the software MultiNA ver. 1.11 (Shimadzu).
The NGS data were analyzed using CRISPResso ver. 1.0.0^{34 (link)} (Fig. 2b) or Cas-Analyzer^{35 (link)} (Fig. 2c, d, Supplementary Fig. 11–13). The parameters used for CRISPResso analysis are described below. The parameters used for Cas-Analyzer were as follows: comparison range, 70; minimum frequency, 0; and WT marker, 5. The exported results of CRISPResso and Cas-Analyzer are described in Supplementary Data 2 and 3, respectively.

Nakade S., Mochida K., Kunii A., Nakamae K., Aida T., Tanaka K., Sakamoto N., Sakuma T, & Yamamoto T. (2018). Biased genome editing using the local accumulation of DSB repair molecules system. Nature Communications, 9, 3270.

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Rapid Transformation and Screening of C. cinerea

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Protoplasts from GFP-expressing C. cinerea were transformed with pCop108_GFP. After 2–3 days incubation, a part of each individual transformed mycelium was picked up and subjected to DNA extraction. The samples were directly dipped in 100 µl TE and heated at 95 °C for 10 min. One µl of the extracts was used as the template for PCR using ExTaq polymerase (Takara) following the manufacturer’s protocol. The PCR products were analyzed by capillary based electrophoresis using MultiNA (Shimazu). The samples were also subjected to direct sequencing using an ABI 3130 Genetic Analyzer (Applied Biosystems), followed by deep sequencing. GFP activity was monitored using a fluorescence stereo microscope Leica M205 FA (Leica Microsystems). The primers used for genotyping are listed in Supplementary Table 1.

Sugano S.S., Suzuki H., Shimokita E., Chiba H., Noji S., Osakabe Y, & Osakabe K. (2017). Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system. Scientific Reports, 7, 1260.

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Genotyping of 5-HTTLPR Polymorphism

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Extraction of genomic DNA from peripheral blood cells (PBCs) and genotyping in the CCSS were previously reported^{28 (link)} and are briefly summarized in the Supplementary Methods. In the ACSS, genomic DNA was extracted from PBCs with a QIAamp DNA Blood Mini QIAcube Kit (Qiagen, Hilden, Germany) using a QIAcube (Qiagen). 5-HTTLPR was amplified with the following primers: FWD 5′-CTTTGCGTTTTCTGTTGCCC−3′ and REV 5′-GGAGGCCAGGAACGATAGGA−3′. PCR amplification was performed in a total volume of 20 µL solutions containing the following compositions: 10 µL of 2× PCR buffer for KOD FX (TOYOBO, Osaka, Japan), 4 µL of 12 mM dNTP mix, 0.6 µL each of 10 µM primers, 2 U of KOD FX (TOYOBO) and 10 ng of genomic DNA. The thermocycling conditions were as follows: an initial cycle of 2 min at 94 °C, followed by 30 cycles of 10 s at 98 °C and 1 min at 68 °C. All PCR amplicons were analyzed by MultiNA (SHIMAZU, Kyoto, Japan) and by bidirectional Sanger sequencing with 5′-GGCGTTGCCGCTCTGAATGC−3′ or 5′-CAGGGCGGGGACCGCAAGGT−3′. In some amplicons, we additionally performed TA cloning using a TOPO cloning kit (Invitrogen, Carlsbad, CA) followed by Sanger sequencing analysis of individual colonies.

Ikegame T., Hidaka Y., Nakachi Y., Murata Y., Watanabe R., Sugawara H., Asai T., Kiyota E., Saito T., Ikeda M., Sasaki T., Hashimoto M., Ishikawa T., Takebayashi M., Iwata N., Kakiuchi C., Kato T., Kasai K., Bundo M, & Iwamoto K. (2021). Identification and functional characterization of the extremely long allele of the serotonin transporter-linked polymorphic region. Translational Psychiatry, 11, 119.

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NKG2D Ligand Expression Analysis

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Total RNA extraction and qPCR were performed as described previously [6 (link)]. Briefly, total RNA was extracted from the cells using the RNeasy^® Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The cDNA was synthesized from 1 µg of the extracted total RNA using 100 pmol of random primers (Takara Bio Inc., Otsu, Japan) and 100 U M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA). The cDNA was then used in the PCR reaction, which was performed using the QIAGEN Multiplex PCR kit (Qiagen, Hilden, Germany). Numerous primer pairs were used to investigate the mRNA expression levels of the following NKG2D ligands: MICA, MICB, ULBP1, ULBP2, and ULBP3. ACTB and ribosomal protein L19 (RPL19) were used as the loading control and the degradation marker, respectively (Bioneer Corporation, Daejeon, Korea). The primer sequences have been reported previously [6 (link)]. The PCR products were separated and quantified with the help of MultiNA (Shimadzu, Tokyo, Japan).

Cho H., Son W.C., Lee Y.S., Youn E.J., Kang C.D., Park Y.S, & Bae J. (2021). Differential Effects of Histone Deacetylases on the Expression of NKG2D Ligands and NK Cell-Mediated Anticancer Immunity in Lung Cancer Cells. Molecules, 26(13), 3952.

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Transcriptome Analysis of Ergot Infection

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The individual ovary halves (up to 12 ovaries halves per ear) collected from each Cp-inoculated ear were pooled if the corresponding ovary half was shown by microscopy to be infected with C. purpurea infection. The half ovaries from one ear formed an RNA replicate. Each ovary half was sectioned into stigma, transmitting and base tissue. Tissue disruption of plant and fungal tissues was carried out using 2 mm RNase-free steel balls (Spheric Transfer). RNA was prepared using the Trizol (Invitrogen) method. RNA was DNase treated (Qiagen) and then cleaned using RNeasy 96-well columns, before quanti cation using nanodrop. RNA integrity was assessed using a Shimadzu MultiNA in order to select 3 of the 5
replicates RNA samples for Illumina TruSeq library preparation. , and reads with low mapping quality (option: view -b -q 5) and PCR duplicates (option: rmdup) were removed [52] Percentage alignment results are provided in Additional le 1 (Table S1). The average proportion of reads removed across all libraries was 0.0093%.

Boyd L.A., Tente E., Ereful N.C., Rodriguez A.C., Grant P.K., O’Sullivan D.M., & Gordon A. (2020). Reprogramming of the Wheat Transcriptome in Response to Infection with Claviceps Purpurea, the Causal Agent of Ergot.

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miRNA Sequencing Library Preparation

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The following reagents were added to 12.5 μL of reverse transcription reaction mixture described in the previous section: 25 μL of PCR mix, 2 μL of miRNA PCR primer, 2 μL of miRNA PCR primer Index, and 8.5 μL of nuclease-free purified water to make the total reaction mixture up to 50 μL. The PCRs were performed under the following conditions: initial denaturation at 98°C for 30 s; followed by 15 cycles of heat denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 15 s; then a final extension at 72°C for 1 min. The sequences of the primers used are shown in Table 2. The PCR products (145 bp to 160 bp) were purified on 6% Novex TBE gels (Life Technologies, Waltham, MA, USA), following the manufacturer’s instructions. The PCR products were evaluated with a microchip based capillary electrophoresis system (MultiNA, Shimadzu, Tokyo, Japan), and the concentrations were measured on a Qubit fluorometer (Thermo Fisher Scientific).

Ochiai C., Miyauchi S., Kudo Y., Naruke Y., Yoneyama S., Tomita K., Dongze L., Chiba Y., Hirata T.I., Ichijo T., Nagai K., Kobayashi S., Yamada S., Hikono H, & Murakami K. (2021). Characterization of microRNA expression in B cells derived from Japanese black cattle naturally infected with bovine leukemia virus by deep sequencing. PLoS ONE, 16(9), e0256588.

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Gene Expression Analysis of Paraquat Exposure

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A total of 72 samples were used for gene expression analysis, corresponding to three groups (controls, 0.3 and 0.4 mM paraquat), with four replicates per group, and each replicate consisting of a pool of six individuals. Care was taken to select samples of similar body size for each pool. Each sample was collected at 4th‐instar, snap frozen with liquid nitrogen, and stored at −80°C. Whole caterpillars were then homogenized in TissueLyser II solution (Qiagen), with 12 × 2 mm ceria‐stabilized zirconium oxide ceramic beads (ZROB20‐RNA, Next Advance) in 500 µl of 90% ethanol at 30 ls⁻¹ for 3 min—where the beads, ethanol, and TissueLyser sample racks were prechilled to −80°C. Samples were then rechilled on dry ice for 1 min and the homogenization process repeated twice. Subsequently, 20–120 µl of combined homogenates from six individuals (totaling 10 mg of tissue per individual) was aliquoted into a pool and thoroughly mixed. Into each pool, 1,200 µl of Lysis Buffer (PureLink^TM RNA Mini Kit) was added, along with 70% ethanol to bring the total to 1,600 µl (achieving a final concentration of 40% ethanol, 50% lysis buffer, and 10% tissues). RNA isolation was conducted following the PureLink^TM RNA Mini Kit protocol. The RNA pellet was re‐suspended with 70 µl RNase free water and quantity/quality checked by Nanodrop (ThermoFisher) and MultiNA (Shimadzu).

Apirajkamol N.(., James B., Gordon K.H., Walsh T.K, & McGaughran A. (2020). Oxidative stress delays development and alters gene expression in the agricultural pest moth, Helicoverpa armigera. Ecology and Evolution, 10(12), 5680-5693.

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Paraquat-Induced Gene Expression in Caterpillars

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A total of 72 samples were used for gene expression analysis, corresponding to three groups (controls, 0.3 mM and 0.4 mM paraquat), with four replicates per group, and each replicate consisting of a pool of six individuals. Care was taken to select samples of similar body size for each pool. Each sample was collected at 4th-instar, snap frozen with liquid nitrogen, and stored at -80°C. Whole caterpillars were then homogenised in TissueLyser II solution (Qiagen), with 12 × 2 mm ceria-stabilised zirconium oxide ceramic beads (ZROB20-RNA, Next Advance) in 500 µl of 90% ethanol at 30 ls -1 for 3 min -where the beads, ethanol and TissueLyser sample racks were pre-chilled to -80°C. Samples were then re-chilled on dry ice for 1 min and the homogenisation process repeated twice. Subsequently, 20-120 µl of combined homogenates from six individuals (totalling 10 mg of tissue per individual) were aliquoted into a pool and thoroughly mixed. Into each pool, 1200 µl of Lysis Buffer (PureLink TM RNA Mini Kit) was added, along with 70% ethanol to bring the total to 1600 µl (achieving a final concentration of 40% ethanol, 50% lysis buffer, and 10% tissues). RNA isolation was conducted following the PureLink TM RNA Mini Kit protocol. The RNA pellet was re-suspended with 70 µl RNase free water and quantity/quality checked by Nanodrop (ThermoFisher) and MultiNA (Shimadzu).

Apirajkamol N., James B., Walsh T., & McGaughran A. (2020). Oxidative stress delays development and alters gene function in the agricultural pest moth, Helicoverpa armigera.

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Amplification and Sequencing of Airborne 16S rDNA

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The V3–V4 region of 16S rDNA in the DNA samples extracted from the aerosol particles was PCR amplified on a PCR thermal cycler SP (Model. TP400, Takara Bio, Kusatsu, Japan), using the primers 341F/R805 (Table 2) [27 (link),28 (link)]. Each 50 µL reaction mixture contained 12 µL of DNA template, Tks Gflex™ DNA polymerase (Code. R060A, Takara Bio, Kusatsu, Japan), and 1 µM of each primer. The amplification protocol consisted of 40 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s. Indexed NGS adapters were attached to each amplified DNA fragment isolated by the PCR products purification kit. Eight sets of forward primers (D501-08, Illumina Inc., San Diego, CA, USA) and two sets of reverse primers (D709-710, Illumina, San Diego, CA, USA) were used. A second PCR amplification was performed using the same protocol as above. Amplified DNA fragments were isolated from each solution using magnetic beads DNA isolation kits (AMPure XP, Beckman Coulter Inc., La Brea, CA, USA), with the quality of the 16S rDNA fragments determined using a capillary electrophoresis device (MultiNA, Shimadzu Co., Kyoto, Japan). DNA concentrations were measured using Qubit dsDNA HS assay kits (Thermo Fisher Scientific Inc., Waltham, MA, USA) and a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

Yin X., Kamba S., Yamamoto K., Ogura A., Wandera E.A., Shah M.M., Seto H., Kondo T., Ichinose Y, & Hasegawa M. (2022). Analysis of Environmental and Pathogenic Bacteria Attached to Aerosol Particles Size-Separated with a Metal Mesh Device. International Journal of Environmental Research and Public Health, 19(9), 5773.

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Genome Assembly of P. aeruginosa PGPR2

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Genomic libraries were generated using 1 μg of genomic DNA using the Ion Xpress Plus fragment library kit (Life technologies, NY, USA). Briefly, genomic DNA was enzymatically sheared and the quality and quantity were analyzed on a bioanalyzer (Multina, Shimadzu, Japan). Adapters were ligated to the sheared DNA fragments and the fragments were size-selected on 2% E-gel (Lifetechnologies). The size selected library (150 to 300 bp) was used for template preparation on an Ion one-touch automated template preparation system using the Ion One-touch Template 200 kit. Template positive ion sphere particles (ISPs) were enriched using the Ion one touch ES system, loaded onto an Ion 316 chip, and sequenced using the Ion sequencing 200 kit on Ion Torrent Personnel Genome machine. The MIRA assembler v 3.4.1 was used to generate a reference genome assembly of P. aeruginosa PGPR2 using P. aeruginosa DK2 genome (NC_018080) as template.
The template sequence was removed after alignment and the unaligned reads were extracted for de novo assembly using MIRA v 3.4.1 [8 ]. The resulting contigs of high quality and appreciable length were added to the assembly. The final draft genome was viewed and edited, when required, using Staden Package version 2.0 [9 (link)].

Illakkiam D., Shankar M., Ponraj P., Rajendhran J, & Gunasekaran P. (2014). Genome Sequencing of a Mung Bean Plant Growth Promoting Strain of P. aeruginosa with Biocontrol Ability. International Journal of Genomics, 2014, 123058.

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