Dig labeled nucleotides

Manufactured by Roche

Sourced in Switzerland

DIG-labeled nucleotides are laboratory reagents used in molecular biology applications. They are modified DNA or RNA nucleotides that incorporate a digoxigenin (DIG) label, which can be detected using specific antibodies. These labeled nucleotides can be used for various purposes, such as DNA or RNA hybridization assays, in situ hybridization, and other detection techniques.

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Lab products found in correlation

Chemiluminescent detection kit, by Thermo Fisher Scientific (3 mentions) 1700 chemiluminescent microarray analyzer, by Thermo Fisher Scientific (3 mentions) Applied biosystems chemiluminescent rt ivt labeling kit, by Thermo Fisher Scientific (3 mentions) Genespring gx 13, by Agilent Technologies (2 mentions) Human genome survey arrays, by Thermo Fisher Scientific (2 mentions) Genechip mouse gene 1.0 st array, by Thermo Fisher Scientific (1 mentions) Genespring gx 12, by Agilent Technologies (1 mentions) Nbt bcip, by Promega (1 mentions) Sp6 polymerase, by Promega (1 mentions) Anti digoxigenin ap, by Roche (1 mentions) Genechip human gene 1.0 st array, by Thermo Fisher Scientific (1 mentions) T7 or sp6 polymerases, by Promega (1 mentions) Isogen, by Qiagen (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Tissuelyser, by Qiagen (1 mentions) Isogen, by Nippon Gene (1 mentions)

8 protocols using dig labeled nucleotides

Chemiluminescent Labeling of cRNA

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The Applied Biosystems Chemiluminescent RTIVT Labeling Kit (Life Technologies, Carlsbad, CA) was used to convert total RNA to digoxigenin (DIG)-labeled cRNA. Double-stranded cDNA was generated using 1 μg of total RNA, transcribed using DIG-labeled nucleotides (Roche Diagnostics, Basel, Switzerland), fragmented, and hybridized for Human Genome Survey Arrays (Life Technologies) following the manufacturer’s instructions. After washing each array, the signal was developed using a chemiluminescent detection kit (Life Technologies). Processed arrays were scanned on a 1700 chemiluminescent microarray analyzer (Life Technologies), and the results were analyzed using GeneSpring GX 13.0 (Agilent Technologies).

Goda H., Nakashiro K.I., Sano Y., Adachi T., Tokuzen N., Kuribayashi N., Hino S, & Uchida D. (2023). KRT13 and UPK1B for differential diagnosis between metastatic lung carcinoma from oral squamous cell carcinoma and lung squamous cell carcinoma. Scientific Reports, 13, 22626.

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Transcriptome Analysis of Pulmonary CD31+ Cells

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CD31-positive cells were assembled from three mice in each group. Total RNA was extracted from pulmonary CD31-positive cells and whole pulmonary cells with Isogen II and collected as one sample in each group. We used 500 ng of total RNA to generate double-stranded cDNA. The cDNA was transcribed with DIG-labeled nucleotides (Roche Diagnostics, Basel, Switzerland), fragmented, and hybridized to a Gene Chip Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. These results were analyzed using the Gene Spring GX 12.1 (Agilent Technologies, Santa Clara, CA) and Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Redwood, CA) software. Functional analysis by IPA identified the biological functions that were most significant to the data set. Fischer's exact test was used to calculate a p-value indicating the probability that each biological function assigned to that data set was due to chance alone. The microarray data are deposited in Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.-gov/geo, Accession number: GSE50088) according to Minimum Information About Microarray Experiment (MIAME) guidelines.

Shikata F., Sakaue T., Nakashiro K.I., Okazaki M., Kurata M., Okamura T., Okura M., Ryugo M., Nakamura Y., Yasugi T., Higashiyama S, & Izutani H. (2014). Pathophysiology of Lung Injury Induced by Common Bile Duct Ligation in Mice. PLoS ONE, 9(4), e94550.

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Whole Mount In Situ Hybridization Protocol for phlda3

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To develop a probe for whole mount in situ hybridization for phlda3, we used RT-PCR analysis to amplify a fragment specific for phlda3 with a forward primer (5’-TGGAGTATAAACGGGGTCTG-3’) and a reverse primer (5’-GCAAAGTGAGGAGTGGAATC-3’). The PCR fragment was subcloned into the pGEM-T easy vector, generating the phlda3-pGEM-T easy plasmid.
The plasmids for making antisense RNA probes for scl, fli1, etsrp, gata1, pu.1, c-myb, runx1, flt4, dab2, notch3, flk1 and cdh5 were described previously by us [28 (link), 40 (link)].
Each plasmid was linearized by restriction digestion and used for making antisense RNA probes for whole mount in situ hybridization using either T7 or SP6 polymerase (Promega) in the presence of DIG-labeled nucleotides (Roche, Mannheim, Germany).
Whole mount in situ hybridization was performed using standard protocols as described by Thisse et al [41 (link)]. The hybridization signals were detected using anti-digoxigenin-AP (Roche Mannheim, Germany) and staining with BCIP/NBT (Promega) as previously described [28 (link), 36 (link), 37 (link), 40 (link), 42 (link)].

Wang X., Li J., Yang Z., Wang L., Li L., Deng W., Zhou J., Wang L., Xu C., Chen Q, & Wang Q.K. (2018). phlda3 overexpression impairs specification of hemangioblasts and vascular development. The FEBS journal, 285(21), 4071-4081.

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Northern Blot Analysis of GFP Transcript

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Northern blot analysis was performed as described previously [28 (link)]. For detection of gfp transcript a 286 bp fragment was PCR-amplified from pBAD2-gfp. RNA probes were derived from in vitro transcription with digoxigenin (DIG) labeled nucleotides (Roche, Mannheim, Germany).

Twittenhoff C., Heroven A.K., Mühlen S., Dersch P, & Narberhaus F. (2020). An RNA thermometer dictates production of a secreted bacterial toxin. PLoS Pathogens, 16(1), e1008184.

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Chemiluminescent Microarray Analysis of Gene Expression

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The Applied Biosystems Chemiluminescent RT-IVT Labeling Kit (Life Technologies, Carlsbad, CA) was used to convert total RNA to digoxigenin (DIG)-labeled cRNA. Double-stranded cDNA was generated from 1 μg total RNA, transcribed using DIG-labeled nucleotides (Roche Diagnostics, Basel, Switzerland), fragmented, and hybridized to Human Genome Survey Arrays (Life Technologies) according to the manufacturer's instructions. After washing each array, the signal was developed using a chemiluminescent detection kit (Life Technologies). Processed arrays were scanned with a 1700 chemiluminescent microarray analyzer (Life Technologies) and the results analyzed using the GeneSpring GX 13.0 (Agilent Technologies). The raw microarray data have been deposited in the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.-gov/geo, experiment number: GSE70604), according to the minimum information about microarray experiment (MIAME) guidelines.

Oka R., Nakashiro K.I., Goda H., Iwamoto K., Tokuzen N, & Hamakawa H. (2015). Annexin A8 is a novel molecular marker for detecting lymph node metastasis in oral squamous cell carcinoma. Oncotarget, 7(4), 4882-4889.

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Transcriptome Analysis of CUL3 Knockdown

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Total RNA was extracted from control or CUL3 siRNA-transfected HUVECs with Isogen II. We used 500 ng of total RNA to generate double-stranded cDNA. The cDNA was transcribed with DIG-labeled nucleotides (Roche), fragmented, and hybridized to a Gene Chip Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions.

Sakaue T., Sakakibara I., Uesugi T., Fujisaki A., Nakashiro K.I., Hamakawa H., Kubota E., Joh T., Imai Y.K., Izutani H, & Higashiyama S. (2017). The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells. Scientific Reports, 7, 42845.

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Antisense RNA Probe Generation for In Situ Hybridization

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For generation of antisense RNA probes for whole-mount in situ hybridization, we first used PCR to amplify a 300–600 bp cDNA fragment for a target gene to be analyzed. The cDNA fragment was subcloned into a pGEM-T easy vector. The pGEM-T easy-derived plasmids were linearized with restriction digestion. Antisense RNA probes were prepared by in vitro transcription from linearized templates by T7 or SP6 polymerases (Promega) in the presence of DIG-labeled nucleotides (Roche, Mannheim). Whole-mount in situ hybridization was performed as described by us previously42 (link)43 (link)44 .

Zhou J., Wang L., Zuo M., Wang X., Ahmed A.S., Chen Q, & Wang Q.K. (2016). Cardiac sodium channel regulator MOG1 regulates cardiac morphogenesis and rhythm. Scientific Reports, 6, 21538.

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Comprehensive RNA Expression Profiling

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Total RNA was extracted by lysing the tissues using ISOGEN (Nippon Gene: Tokyo, Japan), according to the manufacturer’s instructions, and homogenizing them in 0.5 mL of ISOGEN using a Tissue Lyser (Qiagen, Valencia, CA, USA). The integrity of the RNA was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). An Applied Biosystems Chemiluminescent RT-IVT Labeling Kit (Life Technologies, Carlsbad, CA, USA) was used to convert total RNA to digoxigenin (DIG)-labeled cRNA. Double-stranded cDNA was generated from 1 µg of total RNA, transcribed using DIG-labeled nucleotides (Roche Diagnostics, Basel, Switzerland), fragmented, and hybridized to a Human Genome Survey Array containing 32,878 probes (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. After washing each array, the signal was developed using a chemiluminescent detection kit (Life Technologies, Carlsbad, CA, USA). Processed arrays were scanned with a 1700 chemiluminescent microarray analyzer (Life Technologies, Carlsbad, CA, USA) and the results were then analyzed using GeneSpring GX ver. 13.0 (Agilent Technologies, Santa Clara, CA, USA).

Sawatani Y., Komiyama Y., Nakashiro K.I., Uchida D., Fukumoto C., Shimura M., Hasegawa T., Kamimura R., Hitomi-Koide M., Hyodo T, & Kawamata H. (2020). Paclitaxel Potentiates the Anticancer Effect of Cetuximab by Enhancing Antibody-Dependent Cellular Cytotoxicity on Oral Squamous Cell Carcinoma Cells In Vitro. International Journal of Molecular Sciences, 21(17), 6292.

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