highyield t7 biotin16 rna labeling kit  (Jena Bioscience)

Journal: bioRxiv

Article Title: Restoring Shugoshin 1 reduces chromosome errors in human eggs

doi: 10.64898/2026.01.08.698387

Figure Lengend Snippet: (A) Representative immunofluorescence images showing the localization nascent transcripts (EU, green) inside the nucleus (blue) of early GV oocytes. Images show EU levels in negative control (no EU), control (with EU), upon treatment with α-amanitin, and triptolide. Scale bar: 10 µm. (B) Frequency of PSSC with α-amanitin measured by scoring the percentage of eggs with PSSC, similar to levels observed with triptolide . Plots show number of eggs analyzed on top. Statistical significance is measured by Fisher’s exact test, ns = not significant. (C) Representative time-lapse images showing the localization of MajSat RNA in GV and MI oocytes and MII eggs upon microinjection of GV oocytes with UTP-X-Cy3 labeled MajSat (top panel) and control (bottom panel) RNA. MajSat RNA localizes to the pericentromeres during metaphase I and metaphase II stages along with foci present in the cytoplasm. Cy3-labeled control RNA localizes only in the cytoplasm and not on chromosomes. MajSat and control RNA (green), chromosomes (H2B-SNAP, blue) are shown. Scale bars: 10 µm. (D) Co-localization of MajSat RNA and SGO1 observed on metaphase I chromosome spreads upon performing RNA FISH with immunofluorescence. Scale bar: 10 µm.

Article Snippet: The HighYield T7 Cy3 RNA Labelling Kit (Jena Bioscience, #RNT-101-CY3) was used to label amplified sequences with Cy3-labelled UTP (UTP-X-Cy3, 35%), followed by DNA removal using TurboTM DNAse (ThermoFisher).

Techniques: Immunofluorescence, Negative Control, Control, Microinjection, Labeling

Journal: The Journal of General Virology

Article Title: GRHAL1, a novel lncRNA, regulates HIV-1 gene expression by modulating Tat- and Sp1-mediated HIV-1 LTR activation

doi: 10.1099/jgv.0.002288

Figure Lengend Snippet: GRHAL1 interacts with Sp1 and Tat increases this interaction. ( a ) Schematic representation of putative Sp1-binding sites within GRHAL1 including GC boxes, GC-rich hairpin regions, G-quadruplex (g4) motifs and GAGG elements. ( b ) EMSA with 25 ng of Cy5-labelled GRHAL1 probe incubated with indicated concentrations of recombinant Sp1 protein. ( c ) EMSA with 25 ng of Cy5-labelled GRHAL1 probe and 50 ng of recombinant Sp1 protein and indicated competitors. ( d ) EMSA with 25 ng of Cy5-labelled GRHAL1 probe incubated with indicated concentrations of recombinant Sp1 protein. For the competition assay, 500 ng (25-fold excess) unlabeled GRHAL1 was used. ( e ) EMSA was performed with 25 ng of Cy5-labelled GRHAL1 probe incubated with indicated concentrations of recombinant Sp1 protein in binding buffer at 4 °C for 30 min. For super-shift assays, 2 µg of anti-Sp1 antibody (Millipore) was added for an additional 15 min at 4 °C. For all the EMSA assays, protein-bound probe was resolved on 1% agarose gels, then scanned at the 700 nm channel using a LI-COR Odyssey fluorescent scanner. ( f and g ) RIP assay, in which FLAG-tagged Sp1 or a FLAG-tagged non-Sp1 control plasmid was co-expressed with pcDNA-GRHAL1 plasmid in 293 FT cells, followed by isolation of the FLAG–Sp1–GRHAL1 complex from nuclear extracts using anti-FLAG antibody-conjugated magnetic beads. The bound Sp1 protein was pulled out, and Western analysis was performed using antibodies against FLAG peptide with the Jess automated capillary-based Simple Western system ( f ). GRHAL1 RNA fraction pulled out was quantitated by RT-PCR using GRHAL1-specific primers and Taqman probe. ( h and i ) RIP assay in which 293 FT cells were transfected with pcDNA-GRHAL1 plasmid followed by isolation of the endogenous Sp1 complexed with GRHAL1 from nuclear extracts using N-terminus-specific anti-Sp1 antibody conjugated to A/G magnetic beads. The bound Sp1 protein was pulled out, and Western analysis was performed with antibody to Sp1 (Millipore) using the Jess automated capillary-based Simple Western system ( h ). GRHAL1 RNA fraction pulled out was quantitated by RT-PCR using GRHAL1-specific primers and Taqman probe ( i ). (j ) RNA-pulldown assay was carried out by spiking 2.5 µg of JNE with 500 ng of His-tagged recombinant Sp1 and 100 ng of GRHAL1 RNA in the presence or absence of 1 µg of His-tagged recombinant Tat. Complexes of GRHAL1 RNA with His-tagged protein were pulled down using His-Tagged Dynabeads™, and GRHAL1 RNA was quantitated by RT-PCR. For normalization, the eluants were spiked with equal amounts of RSV particles, then the RNA was isolated and subject to RT-PCR using GRHAL1- and RSV-specific primers.

Article Snippet: Cy5-labelled GRHAL1 RNA probe was generated from 1 μg of GRHAL1 DNA PCR product via a T7-polymerase-mediated in vitro transcription assay by using a HighYield T7 Cy5 RNA labelling kit (UTP-based) (Cat. # RNT-101-CY5, Jena Bioscience).

Techniques: Binding Assay, Incubation, Recombinant, Competitive Binding Assay, Control, Plasmid Preparation, Isolation, Magnetic Beads, Western Blot, Simple Western, Reverse Transcription Polymerase Chain Reaction, Transfection