Easy dilution

Manufactured by Takara Bio

Sourced in China, Japan

EASY Dilution is a lab equipment product designed for the dilution of samples. It provides a simple and efficient way to perform serial dilutions, a common technique in various laboratory workflows.

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Lab products found in correlation

Primescript rt reagent kit, by Takara Bio (2 mentions) Tb green premix ex taq 2, by Takara Bio (2 mentions) Cfx connect real time pcr detection system, by Bio-Rad (1 mentions) Mir xtm mirna first strand synthesis kit, by Takara Bio (1 mentions) Sybr green real time pcr master mix, by Takara Bio (1 mentions) Thermal cycler dice real time system 3, by Takara Bio (1 mentions) Tb green premix ex taqtm 2, by Takara Bio (1 mentions) Rnaiso, by Takara Bio (1 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions) Nanodrop, by Thermo Fisher Scientific (1 mentions) Abi 7500, by Thermo Fisher Scientific (1 mentions) Sybr green, by Takara Bio (1 mentions) Rox dye 2, by Takara Bio (1 mentions) Lightcycler 480 system, by Roche (1 mentions) Nanodrop 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions) Hipure plant rna kit, by Magen Biotechnology Co (1 mentions) Superscript 4 vilo master mix, by Thermo Fisher Scientific (1 mentions) Lightcycler 96, by Roche (1 mentions) Sybr premix ex taq, by Takara Bio (1 mentions)

Close spelling variants of this product

EASY Dilution buffer (5 citations) EASY dilution solution (3 citations) EASY Dilution for Real Time PCR (2 citations)

18 protocols using easy dilution

Validation of miRNA Sequencing Data

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To validate the sequencing data, eight randomly selected miRNAs (including six known and two novel miRNAs) were validated by stem-loop quantitative real-time reverse transcription PCR (qRT-PCR). Total RNA (5 ng) was reverse transcribed into cDNA using the Mir-X^TM miRNA First-Strand Synthesis Kit (TaKaRa, Dalian, China) according to the user’s manual. To detect the expression patterns of target genes of differentially expressed miRNAs, cDNA was synthesized using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). The cDNA products were diluted 1:5 (v/v) with EASY Dilution (TaKaRa, Dalian, China) and stored at -20°C. Primers used in this study are shown in Supplementary Table S8.
Quantitative real-time reverse transcription PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Singapore) in a volume of 10 μL including 5 μL of SYBR Green Real Time PCR Master Mix (TaKaRa, Dalian, China), 0.2 μL each of forward and reverse primer, 3.8 μL of RNase-Free dH₂O, and 0.8 μL of cDNA. U6 snRNA was used as the reference for miRNA and target gene candidates (Guo et al., 2016a (link)). All reactions were performed in triplicate, and the relative expression level was normalized by 2^-ΔΔC_T method.

Zhong T., Hu J., Xiao P., Zhan S., Wang L., Guo J., Li L., Zhang H, & Niu L. (2017). Identification and Characterization of MicroRNAs in the Goat (Capra hircus) Rumen during Embryonic Development. Frontiers in Genetics, 8, 163.

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Quantitative RNA Expression Analysis

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Total RNA was extracted from cells or tissues by the phenol chloroform method using RNAiso (Takara Bio, Inc). Using the PrimeScript RT reagent kit (Takara Bio, Inc), cDNA was synthesized from 1 μg of total RNA after removal of genomic DNA. Random primers were used for reverse transcription reactions other than strand specific. qPCR was performed using Thermal Cycler Dice Real Time System III (Takara Bio, Inc) according to recommended cycling parameters using 1 μl cDNA diluted fivefold with EASY Dilution (Takara Bio, Inc) and TB Green Premix Ex Taq TMII (Takara Bio, Inc). Gene expression levels were normalized to GAPDH by the ΔΔCT method. Primer information for chain-specific RT and primer walking, and for other qRT–PCR, is detailed in Tables S1 and S2, respectively.

Akimoto M., Susa T., Okudaira N., Hisaki H., Iizuka M., Okinaga H., Okazaki T, & Tamamori-Adachi M. (2022). A novel LncRNA PTH-AS upregulates interferon-related DNA damage resistance signature genes and promotes metastasis in human breast cancer xenografts. The Journal of Biological Chemistry, 298(7), 102065.

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RPA Assay Sensitivity and Specificity

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To evaluate the sensitivity of the RPA assay, a recombinant plasmid constructed with a positive specimen (GenBank Accession No. Z67970.1) was used to determine the limit. The plasmid DNA standard was quantified via the DeNovix DS-11 Spectrophotometer and diluted with serial 10-fold ranging from 1 ng to 0.1 fg in Easy dilution (TAKARA, Beijing, China). These templates were then subjected to the RPA assay in the conditions described above.
The specificity of RPA assay was assessed against other common infecting viruses in avian including Avian leukosis virus (DQ115805), Avian influenza virus (MF581307), Newcastle disease virus (MF581294), Tembusu virus (KJ740746), and Goose parvovirus (MF581304).

Zhang J., Liu J., An D., Fan Y., Cheng Z., Tang Y, & Diao Y. (2020). A novel recombinase polymerase amplification assay for rapid detection of epidemic fowl adenovirus. Poultry Science, 99(12), 6446-6453.

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Quantitative Real-Time PCR Protocol

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Total RNA was reverse-transcribed using PrimseScript^™ RT Master Mix (Takara Bio Inc.), followed by 10-fold dilution with EASY Dilution (Takara Bio Inc.). The real-time PCR were performed using TB Green^® Premix Ex Taq^™ II (Takara Bio Inc.) with the 7500 Fast real-time PCR system (Thermo Fisher Scientific Inc.). The thermal cycle condition was an initial step of 10 s at 95 °C, followed by 40 cycles of 3 s at 95 °C and 20 s at 62.5 °C. Gene expression changes were calculated using the 2^−ΔΔCt method. Primers used for the qRT-PCR are shown in Supplementary Table 1.

Suzuki T., Furuhata E., Maeda S., Kishima M., Miyajima Y., Tanaka Y., Lim J., Nishimura H., Nakanishi Y., Shojima A, & Suzuki H. (2022). GATA6 is predicted to regulate DNA methylation in an in vitro model of human hepatocyte differentiation. Communications Biology, 5, 414.

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Construction and Validation of a Multi-virus Plasmid

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A synthetic gene fragment (PEDV-M-TGEV-N-PoRVA-NSP3) containing partial sequences of PEDV M gene, TGEV N gene and PoRVA NSP3 gene was constructed in Sangon Biotech Co., Ltd. (Shanghai, China). This fragment was inserted into the pUC57 cloning vector, forming a standard plasmid (pUC57-PEDV M & TGEV N & PoRVA NSP3) for subsequent detection (Figure 2). The sequence of the synthetic gene fragment is shown in Supplementary Table S1. The plasmids were quantified by ultraviolet absorbance at 260 and 280 nm wavelengths using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and their copy number was calculated based on the size of the standard plasmid template using the following formula: copies/μL = (A260 (ng/μL) × 10⁻⁹ × 6.02 × 10²³)/(DNA length × 650). The standard plasmids were serially diluted 10-fold to a concentration gradient of 10⁸–10⁰ copies/μL with EASY Dilution (TaKaRa, China, Dalian).

Luo T., Li K., Li C., Xia C, & Gao C. (2024). Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A. Frontiers in Microbiology, 15, 1390328.

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Quantifying cfDNA Integrity by ALU PCR

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The integrity of cfDNA was determined by qPCR as previously reported 12 (link). Two primer sets (Sangon Biotech) were used to amplify ALU sequence: the primer set for 115 bp ALU amplicon was: forward 5'-CCTGAGGTCAGGAGTTCGAG-3' and reverse 5'-CCCGAGTAGCTGG GATTACA-3'; the primer set for 247 bp ALU amplicon was: forward 5'-GTGGCTCACGCCTGTAATC-3' and reverse 5'-CAGGCTGGAGTGCAGTGG-3'. The qPCR was done in a final volume of 20 μL on the ABI 7500 (Applied Biosystems) in triplicate and each reaction mixture contained 2 μL cfDNA template, 0.5 μL of each forward and reverse primer (10 μM, ALU115 or ALU247), 10 μL 2x SYBR Green (Takara), 0.5 μL Rox Dye II, and 6.5 μL RNase-free water. The reaction condition was 95 ^oC for 10 s, followed by 40 cycles of 95 ^oC for 5 s, and annealing at 60 ^oC for 34 s. The 115-bp ALU amplicon represented the total amount of DNA fragments including both short and long copies whereas the 247-bp ALU amplicon only reflected the amount of long DNA fragments. The amount of ALU 115 and ALU 247 DNA fragments was determined by comparing the CT values of each sample against the calibration curve created by performing qPCR with serial diluted cDNA (100 ng to 0.01 pg; EASY Dilution, Takara). cfDNA integrity was calculated as the relation of ALU 247 to ALU 115 according to the methods of Umetani et al 12 (link).

Huang A., Zhang X., Zhou S.L., Cao Y., Huang X.W., Fan J., Yang X.R, & Zhou J. (2016). Plasma Circulating Cell-free DNA Integrity as a Promising Biomarker for Diagnosis and Surveillance in Patients with Hepatocellular Carcinoma. Journal of Cancer, 7(13), 1798-1803.

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Comparative Evaluation of RT-LAMP Assays

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The detection limit of the RT-LAMP test with the P25 primer set was determined using
10-fold serial dilutions of viral RNAs and synthesized DNAs of TK and KZ strains with Easy
Dilution (Takara Bio Inc.). The detection limit of the RT-LAMP test was compared with that
of the RT-LAMP test with the Aebischer’s primer set, and with that of the real-time RT-PCR
(SYBR) using Vilcek original primer set [11 (link)], which
is currently used at the Livestock Hygiene Service Centers in Japan.

MUNGTHONG K., KHAING S.T., OTSUBO T., HATANAKA C., YONEYAMA S., HISAMATSU S., MURAKAMI H, & TSUKAMOTO K. (2021). Broad detection and quick differentiation of bovine viral diarrhea viruses 1 and 2 by a reverse transcription loop-mediated isothermal amplification test. The Journal of Veterinary Medical Science, 83(8), 1321-1329.

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MADS-box Gene Expression Analysis

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To analyze the expression profiles of these 11 MADS-box genes in different organs and under different abiotic stress conditions, quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a LightCycler 480 System (Roche, Germany). For the qRT-PCR, the cDNA was diluted 1:50 with EASY Dilution (Takara, Japan). Each reaction contained 20 μl (10 μl SYBR Premix Ex Taq II (2×), 2.0 μl cDNA, 0.8 μM of each primer and 6.4 μl ddH₂O). The following program was performed: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 68 °C for 20 s. Three biological and three technical replicates were executed with LcACTIN as the internal control. The results of the qRT-PCR were analyzed with the 2^-ΔΔCt method, and all primers used in this study are listed in Additional files 1: Table S1.

Jia J., Zhao P., Cheng L., Yuan G., Yang W., Liu S., Chen S., Qi D., Liu G, & Li X. (2018). MADS-box family genes in sheepgrass and their involvement in abiotic stress responses. BMC Plant Biology, 18, 42.

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Certified SARS-CoV-2 RNA Standard Preparation

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Certified reference material of SARS-CoV-2 genome RNA was purchased from the National Reference Material Resource Sharing Platform (http://www.ncrm.org.cn, GBW(E)091099) and the copies number of ORF1ab, E and N genes per reaction were calculated with the instructions. ORF1ab was the largest and the most conserved gene regions within SARS-CoV-2 genome, so we chose ORF1ab as the standard in our studies (Hu et al., 2021 (link)). Different gradient dilutions were prepared with EASY Dilution (9160; Takara, Dalian, China), and a panel of SARS-CoV-2 RNA standards ranging from 448 to 4 copies (ORF1ab gene) per reaction was used for further studies (Table 1).

Yang Z., Liu N.Y., Zhu Z., Xiao M., Zhong S., Xue Q., Nie L, & Zhao J. (2022). Rapid and convenient detection of SARS-CoV-2 using a colorimetric triple-target reverse transcription loop-mediated isothermal amplification method. PeerJ, 10, e14121.

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Sensitive Multiplex Detection of GM Maize

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The genomic DNAs of all plant materials were extracted and purified using a mini-plant genomic DNA extraction kit (Shanghai Bio-ful Biotech Co., Ltd., Shanghai, China). The DNA concentration and quality were estimated using a NanoDrop 1000 UV-Vis spectrophotometer (NanoDrop Technologies, LLC, Wilmington, DE). To test the 4-plex sensitivity, a series of DNA solution were obtained by mixing the GM maize flour with non-GM maize flour. The final relative content was 10%, 5%, 1% and 0.5% (w/w) for each of the four GM maizes, respectively. Event-specific genes of the four GM maize were used for the construction of standard plasmids as calibrators; plasmids were serially diluted from 10⁷ to 10³ and amplified for quantitative detection. Easy dilution (TaKaRa biotechnology Co., Ltd, Dalian, China) was used to dilute the standard plasmids to avoid DNA loss due to adsorption to the tube walls.

Du Y., Zhao X., Zhao B., Xu Y., Shi W., Ren F., Wu Y., Hu R., Fan X., Zhang Q., Zhang X., Zhang W., Wu W., Shi B., Zhao H, & Zhao K. (2019). A novel emulsion PCR method coupled with fluorescence spectrophotometry for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets. Scientific Reports, 9, 184.

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