Isogen ls reagent

Manufactured by Nippon Gene

Sourced in Japan

ISOGEN-LS is a reagent used for the extraction and purification of total RNA from various biological samples. It is a monophasic solution containing phenol and guanidinium thiocyanate, which facilitates the isolation of high-quality RNA.

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Lab products found in correlation

Qiashredder, by Qiagen (1 mentions) Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions) Isogen reagent, by Nippon Gene (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Qiaamp viral rna mini kit, by Qiagen (1 mentions) Lymphoprep, by Abbott (1 mentions) Peg ittm virus precipitation solution, by System Biosciences (1 mentions) Rnase free dnase, by Takara Bio (1 mentions) Primescript reverse transcriptase, by Takara Bio (1 mentions)

6 protocols using isogen ls reagent

RNA Extraction from Diverse Samples

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Total RNA samples were extracted from effusions, supernatants of phosphate-buffered saline-homogenised faecal and rectal swab samples, serum, plasma, and a vitreous humor sample from an eye
using a QIAamp^® Viral RNA Mini Kit (QIAGEN, Hilden, Germany) or ISOGEN-LS reagent (NIPPON GENE, Tokyo, Japan). RNA samples of whole blood were extracted using ISOGEN-LS reagent.
In some cases, erythrocytes were lysed using 0.2% sodium chloride to isolate leukocytes, and their RNA was extracted using an RNeasy^® Mini Kit (QIAGEN) in combination with a QIA
shredder (QIAGEN). Tissues were homogenised in ISOGEN reagent (NIPPON GENE) using a TissueRuptor with TissueRuptor disposable probes (QIAGEN). cDNAs were synthesised using a PrimeScript™ 1st
Strand cDNA Synthesis Kit (Takara Bio, Kusatsu, Japan). All reagents and kits were used according to the manufacturers’ instructions.

OGUMA K., OHNO M., YOSHIDA M, & SENTSUI H. (2018). Mutation of the S and 3c genes in genomes of feline coronaviruses. The Journal of Veterinary Medical Science, 80(7), 1094-1100.

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Extracting Circulating Extracellular RNA

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A total of 6 ml EDTA-preserved blood samples were centrifuged at 650 × g for 20 min at room temperature. Plasma was separated from the samples and was filtered through a 0.45 µM Minisart^® syringe filter membrane (Merck Millipore, Darmstadt, Germany). A total of 200 µl plasma was diluted with 50 µl RNase free water and 1 µl glycogen (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) prior to homogenization with 750 µl Isogen-LS reagent (Nippon Gene Co., Ltd., Tokyo, Japan) by vortexing and pipetting. Synthetic cel-miR-39 RNA (1 fmol; Qiagen GmbH, Hilden, Germany) was spiked into the denatured sample solution. Chloroform (200 µl) was subsequently added, and the suspension was shaken vigorously. The samples were then centrifuged at 18,000 g, 4°C for 15 min, and the RNA in the aqueous phase was precipitated by adding 400 µl isopropanol for 10 min at room temperature. RNA pellets were washed with 1 ml ethanol (70%) and resolubilized in 15 µl RNase-free water.

Leecharoenkiat K., Tanaka Y., Harada Y., Chaichompoo P., Sarakul O., Abe Y., Smith D.R., Fucharoen S., Svasti S, & Umemura T. (2017). Plasma microRNA-451 as a novel hemolytic marker for β0-thalassemia/HbE disease. Molecular Medicine Reports, 15(5), 2495-2502.

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Isolation of Packed Red Cells from Thalassemia Patients

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The packed red cells were separated from the same cohort, including 9 normal subjects, 9 mild and 9 severe β⁰-thalassemia/HbE patients. Following separating plasma from whole blood samples by centrifugation at 650 × g for 20 min at room temperature, the remaining fraction was further used to separate packed red cells by density gradient centrifugation using Lymphoprep (density 1.077±0.001 g/ml; Alere Technologies GmbH, Jena, Germany) to eliminate mononuclear cells. A total of 100 µl of packed red cells were separated and homogenized with 750 µl Isogen-LS reagent (Nippon Gene Co., Ltd.). A total of 200 µl chloroform was added, and the suspension was shaken vigorously. The homogenized samples were centrifuged at 18,000 g for 15 min before RNA in the aqueous phase was separated into a fresh tube and precipitated by incubating in 400 µl isopropanol for 10 min at room temperature. RNA pellets were washed with 1 ml ethanol (70%) and resolubilized in 15 µl RNase-free water.

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Gastric Cancer Biospecimen Collection

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PB samples were obtained from 6 healthy volunteers and the 168 patients with gastric cancer prior to surgery. PB samples (10 ml) were taken and mixed with 4 ml ISOGEN-LS reagent (Nippon Gene Co., Ltd., Toyama, Japan) per 1 ml whole blood. The sample was stored for 5 min at room temperature, then snap-frozen immediately in liquid nitrogen and stored at −80°C until required for RNA extraction.
BM samples were obtained from 160 patients with gastric cancer under general anesthesia prior to surgery. BM samples were taken from the sternum using a 15-gauge needle. Since there was a possibility of aspirating skin with the BM samples, the first 1 ml of the sample was discarded, and another syringe was used to aspirate 20–30 ml of BM. ISOGEN-LS reagent was then added as previous, and the BM samples were subsequently processed in the same manner as the PB samples.
A total of 10 tumor and corresponding normal 4-µm thick formalin-fixed paraffin-embedded tissue section samples from the patients who underwent surgery were also obtained and used for immunohistochemical analysis. A total of 6 autopsy bone marrow samples were collected, 3 from patients who succumbed to advanced gastric cancer, and 3 from pneumonia. Formalin-fixed paraffin-embedded 4-µm tissue sections were made from these bone marrow samples.

Sudo T., Takahashi Y., Sawada G., Uchi R., Mimori K, & Akagi Y. (2017). Significance of CD47 expression in gastric cancer. Oncology Letters, 14(1), 801-809.

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Isolation and Purification of HIV-1 mRNAs

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Total cellular mRNAs expressed in human T cell line persistently infected with HIV-1IIIB strain, Molt-4/IIIB cells23 (link) were isolated by using Isogen-LS reagent (Isogen, Nippon Gene, Toyama, Japan) followed by oligo-(dT) column purification (Oligotex™-dT30, Takara & Clontech Laboratories Inc. Japan). For preparation of HIV-1 genomic RNAs in virus particles, culture supernatant of Molt-4/IIIB cells was harvested and filtered through 0.45-μ-pore-size filters. Then, virus particles in the culture supernatant were precipitated by using PEG-it system according to the manufacture’s protocol (PEG-itTM virus precipitation solution, System Biosciences, CA, USA). Total mRNA from the virus particles was similarly isolated. To remove the cap moiety of mRNA, each isolated mRNA fraction was treated with Tobacco Acid Pyrophosphatase (TAP, Nippon Gene, Toyama, Japan) in the reaction buffer containing 200 units of TAP, 50 mM sodium acetate, 5 mM EDTA and 10 mM 2-mercaptoethanol for 37 °C for 60 min.

Masuda T., Sato Y., Huang Y.L., Koi S., Takahata T., Hasegawa A., Kawai G, & Kannagi M. (2015). Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA. Scientific Reports, 5, 17680.

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RT-PCR Analysis of Porphyromonas gingivalis

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RT-PCR analysis was carried out as previously described [31]. Briefly, total RNA was isolated from P. gingivalis using ISOGEN-LS reagent (Nippon Gene, Tokyo, Japan) in accordance with the manufacturer’s instructions. Total RNA samples were treated with RNase-free DNase (TaKaRa Bio, Kusatsu, Japan) to remove traces of chromosomal DNA. Total RNA was reverse-transcribed with random hexadeoxyribonucleotide primers (TaKaRa Bio) using PrimeScript Reverse Transcriptase (TaKaRa Bio) according to the manufacturer’s instructions. Gene-specific primers used for RT-PCR are listed in the Supplemental Table. Reaction mixtures without reverse transcriptase served as negative controls to evaluate the presence of contaminating genomic DNA in samples.

Yoshida Y., Sato M., Nonaka T., Hasegawa Y, & Kezuka Y. (2019). Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis. Journal of Oral Microbiology, 11(1), 1588086.

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