Digoxigenin labeling kit

Manufactured by Roche

Sourced in Germany

The Digoxigenin labeling kit is a laboratory product designed for the detection and analysis of nucleic acid targets. It allows for the incorporation of the hapten digoxigenin into DNA or RNA samples, enabling their subsequent identification and visualization through immunological methods. The kit provides the necessary reagents and protocols for the labeling process, facilitating the study of gene expression, DNA hybridization, and other molecular biology applications.

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Lab products found in correlation

Pgem t easy vector, by Promega (4 mentions) T7 rna polymerase, by Promega (2 mentions) Bm purple, by Roche (1 mentions) Dx41 microscope, by Olympus (1 mentions) Anti digoxigenin ap, by Roche (1 mentions) Messagemachine, by Thermo Fisher Scientific (1 mentions) Axio zoom v16, by Zeiss (1 mentions) Axiocam 506, by Zeiss (1 mentions) Zen pro software, by Zeiss (1 mentions) Ds ri1 camera, by Nikon (1 mentions) Bx60 microscope, by Nikon (1 mentions) Nbt bcip, by Roche (1 mentions) Rneasy minelute cleanup kit, by Qiagen (1 mentions) Rm2255 rotary microtome, by Leica (1 mentions)

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9 protocols using digoxigenin labeling kit

In Situ Hybridization of Protein-Coding Genes

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For the in situ hybridization experiments, we collected siliques with embryos at different stages from each genotype. To prepare probes, we amplified fragments of protein-coding genes by PCR from Ler cDNA and cloned them into pGEM-T Easy vectors (Promega, catalog no. A1360) for in vitro transcription using the Digoxigenin Labeling Kit (Roche, catalog no. 11277073910). Antisense and sense probes were synthesized using SP6 (Promega, catalog no. P1085) or T7 RNA polymerase (Promega, catalog no. P2075). The long probes were then hydrolyzed to an average length of 150 bp with carbonate buffer (2× buffer: 80 mM NaHCO₃, 120 mM Na₂CO₃) and resuspended in 50% formamide at the desired concentration. The miR166 locked nucleic acid (LNA) probe (zma-miR166a) was obtained from the EXIQON company with 5′ end labeling of digoxigenin: /5′Dig/GGG GAA TGA AGC CTG GTC CGA. Primers for amplifying hybridization probes are listed in Supplemental Table 6.
In situ hybridization experiments were performed as previously described (Zhang et al., 2017 (link)). For the miR166 LNA probe, the hybridization and washing were performed at 60°C and staining at 4°C, as previously described (Liu et al., 2009 (link)). For other probes, the hybridization and washing were performed at 55°C and staining at room temperature.

Du F., Gong W., Boscá S., Tucker M., Vaucheret H, & Laux T. (2019). Dose-Dependent AGO1-Mediated Inhibition of the miRNA165/166 Pathway Modulates Stem Cell Maintenance in Arabidopsis Shoot Apical Meristem. Plant Communications, 1(1), 100002.

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In Situ Hybridization for Ascl3 in Mouse Olfactory Epithelium

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Ascl3 sense and antisense riboprobes were described previously29 (link) and generated using the digoxigenin-labeling kit (Roche) followed by incubation with Anti-Digoxigenin-AP (Roche). The signal was detected using BM Purple (Roche). All frozen sections of mouse olfactory epithelium used were 10 μm. Images were taken using an Olympus DX41 microscope with a DP71 camera, analyzed on DP-BSW-V3.2 software and processed using Adobe Photoshop (Olympus America Inc). Figures were assembled using Adobe Photoshop.

Weng P.L., Vinjamuri M, & Ovitt C.E. (2016). Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium. Scientific Reports, 6, 38199.

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In Situ Hybridization with Digoxigenin Probe

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eIF3i probe was synthesized with a digoxigenin labeling kit (Roche Applied Science). Whole-mount in situ hybridizations were performed as previously described [26 (link)].

Zhang Y., Wang P., Zhang Q., Yao X., Zhao L., Liu Y., Liu X., Tao R., Yu C., Li Y., Song X, & Yao S. (2017). eIF3i activity is critical for endothelial cells in tumor induced angiogenesis through regulating VEGFR and ERK translation. Oncotarget, 8(12), 19968-19979.

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Zebrafish Embryonic Development Protocols

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Wild-type zebrafish (strain AB) were maintained at 28.5 °C using standard protocols. Procedures for animal staging and injection were performed as described^{37 (link),38 (link),39 (link),40 (link)}. Synthetic RNAs were transcribed in vitro using pCS2-Twa1a, Twa1b or Twa1a-ΔCRA vectors with MessageMachine (Ambion). All morpholinos (MOs) were purchased from Gene Tools. The following targeting sequences were used: Twa1a: 5′-CTGGCTTTTCAGCATAACTCATCAT-3′ Twa1b: 5′-CAGGCTTTTCTGAATAGCTCATCACA-3′ β-catenin 2: 5′-AGCCATCGTTGCGTCAATCCTTTAG-3′ standard control: 5′-CCTCTTACCTCAGTTACAATTTATA-3′. The doses of each MO and mRNA for injection were 6 ng and 15 pg, respectively.
For in situ hybridization, the sequences of probes (boz, chordin, eve1, gata2, gsc and otx2) were amplified from zebrafish cDNAs and cloned into pCS2+ vectors^{37 (link),38 (link),39 (link),40 (link)}. All constructs were confirmed by DNA sequencing. The probes were labeled using the Digoxigenin labeling kit (Roche). Whole-mount in situ hybridization was carried out as described previously^{37 (link),38 (link),39 (link),40 (link)}.

Lu Y., Xie S., Zhang W., Zhang C., Gao C., Sun Q., Cai Y., Xu Z., Xiao M., Xu Y., Huang X., Wu X., Liu W., Wang F., Kang Y, & Zhou T. (2017). Twa1/Gid8 is a β-catenin nuclear retention factor in Wnt signaling and colorectal tumorigenesis. Cell Research, 27(12), 1422-1440.

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Whole-Mount In Situ Hybridization of Embryos

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Embryos at different development stages were collected and fixed using 4% paraformaldehyde in PBS for 2 h at 4°C, dehydrated with an increasing scale of methanol/PBS and transferred in 100% methanol for storage at −20°C. The ORF of the four co-orthologs were amplified (primers in Supplementary Table S1) and cloned in pGem-T-Easy vector (Promega). We synthesized labeled sense and anti-sense probes by T7 and SP6-driven transcription with the digoxigenin labeling kit (ROCHE) according to manufacturer’s instructions. WISH was performed according to a standard protocol (Thisse and Thisse, 2008 (link)). Probe hybridization was revealed by overnight incubation with an anti-DIG antibody conjugated with alkaline phosphatase (1:10,000) at 4°C and staining by NBT/BCIP solution. WISH images were captured using a Zeiss Axio Zoom V16 equipped with Zeiss Axiocam 506 color digital camera and processed using Zen Pro software from Zeiss.

Mignani L., Zizioli D., Borsani G., Monti E, & Finazzi D. (2020). The Downregulation of c19orf12 Negatively Affects Neuronal and Musculature Development in Zebrafish Embryos. Frontiers in Cell and Developmental Biology, 8, 596069.

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Characterizing SlWOX1 Expression via In Situ Hybridization

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A 773-bp coding sequence of SlWOX1 was PCR-amplified from M82 cDNA and cloned into pGEM-T Easy vectors (Promega, Cat. no. A1360) for in vitro transcription using a digoxigenin labeling kit (Roche, Cat. no. 11277073910) . Anti-sense and sense probes were synthetized using SP6 (Promega, Cat. no. P1085) and T7 RNA polymerase (Promega, Cat. no. P2075), respectively. The long probes were then hydrolyzed to an average length of 150 bp and resuspended in 50% formamide at the desired concentrations. Primers for amplifying probes are listed in Table S1.
In situ hybridization experiments were performed as previously described (Zhang et al., 2017) . The paraffin sections were 8 lm thick. Both the hybridization and washing were performed at 55°C, and staining was conducted at room temperature. Optical photographs of in situ hybridization sections were taken with an Olympus BX60 microscope equipped with a Nikon DS-Ri1 camera.

Du F., Mo Y., Israeli A., Wang Q., Yifhar T., Ori N, & Jiao Y. (2020). Leaflet initiation and blade expansion are separable in compound leaf development. The Plant journal : for cell and molecular biology, 104(4).

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Non-radioactive in situ Hybridization on Cryosections

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Non-radioactive in situ hybridization on cryosections and preparation of digoxigenin-labeled probes were carried out as described by Ernsberger et al. (1997 (link)). Riboprobes were labeled with a digoxigenin labeling kit (Roche, Mannheim, Germany) and revealed by BCIP/NBT (Roche). Gli1, Gli2, Gli3 and Ptch1 ISH probes were kindly provided by Dr. Alexandra L.Joyner (Howard Huges Medical Institute, Skirball Institute of Biomolecular Medicine, New York, USA; Platt et al. 1997 (link)) and the Neurofilament ISH probe was provided by Dr. Katrin Huber (Department of Medicine, University of Fribourg, Fribourg, Switzerland; Huber et al. 2002 (link)). Subsequently, sections were processed for immunohistochemistry as described below.

Feuerstein M., Chleilat E., Khakipoor S., Michailidis K., Ophoven C, & Roussa E. (2017). Expression patterns of key Sonic Hedgehog signaling pathway components in the developing and adult mouse midbrain and in the MN9D cell line. Cell and Tissue Research, 370(2), 211-225.

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Synthesis of RNA in situ Hybridization Probes

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RNA in situ hybridization probes were synthesized as previously described (Smith, 2018 (link); Smith et al., 2016 (link)), using the T7/T3 Riboprobe Combination System kit (Promega) and the Digoxigenin labeling kit (Roche). Both anti-sense and sense probes were made for each gene, using T7 or T3 for each, depending on the orientation of each gene insertion in the TOPO vector (identified by sequencing results). Probes were cleaned using an RNEasy MinElute Cleanup Kit (Qiagen) and diluted in RNAse-free water and kept at −80C. Final probe concentration for in situ reactions was 0.5 μg/mL, as recommended in (Smith, 2018 (link)).

Heikes K.L., Game M., Smith F.W, & Goldstein B. (2023). The Embryonic Origin of Primordial Germ Cells in the Tardigrade Hypsibius exemplaris. bioRxiv.

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Generating Probes for Gene Expression

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The probe for ARF6 was generated through amplifying nucleotides +1180 to +2370 of the ARF6 coding sequence with primers oFD119 and oFD120. The probe of ARF8 was generated through amplifying nucleotides +1138 to +2099 of the ARF8 coding sequence with primers oFD121 and oFD122. The probe of DWF4 was generated through amplifying nucleotides +1 to +1068 of the DWF4 coding sequence with primers oFD123 and oFD124. The PCR fragments were cloned into the pGEM-T Easy vector (Promega) for in vitro transcription using the Digoxigenin Labeling Kit (Roche). The long probes were then hydrolyzed to an average length of 150 bp and resuspended in 50% formamide at the desired concentrations. Primers for amplifying probes are listed in Supplemental Table 1.
Nonradioactive in situ hybridization was performed on 8 mm paraffin sections cut with a Leica RM2255 rotary microtome as described previously (Zhang et al., 2017) . Both the hybridization and washing processes were performed at 55 C, with signal development at room temperature.

Xiong Y., Wu B., Du F., Guo X., Tian C., Hu J., Lü S., Long M., Zhang L., Wang Y, & Jiao Y. (2021). A crosstalk between auxin and brassinosteroid regulates leaf shape by modulating growth anisotropy. Molecular plant, 14(6).

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