high yield t7 arca mrna synthesis kit  (Jena Bioscience)

Journal: Human Reproduction (Oxford, England)

Article Title: A homozygous variant in HFM1 causes preimplantation embryo developmental arrest by disrupting zygotic genome activation

doi: 10.1093/humrep/deaf238

Figure Lengend Snippet: Identification of a novel HFM1 mutation. ( A ) A patient with an HFM1 gene mutation was identified in a Chinese family. The female patient (II-3) of the family was diagnosed with infertility. The double line represents infertility; the square represents a male member; the circle represents a female member; and the all-black solid symbol represents an infertile patient with a homozygous mutation. The semi-black solid symbol expresses the carrier of the gene mutation, and the hollow indicates that the gene mutation is not carried. WT, wild type; nM _ 001017975.6: c.2680 + 3 _ 2680 + 4del, indicating the mutation position of the gene. ( B ) Sanger sequencing peak map of the patient’s family. The black arrow represents the base before the mutant base, and the red box represents the mutant base and the affected base sequence behind it. ( C ) This figure represents the structural diagram of HFM1 at the gene, mRNA, and protein levels. The red arrow indicates the mutation site of HFM1 in the patient. The schematic gene structure and mRNA structure diagram data were obtained from the NCBI database, and the protein structure diagram data were obtained from the UniProt database.

Article Snippet: The target gene fragments were amplified by PCR, and in vitro transcription of capped mRNA was performed using the HiScribe ® T7 ARCA mRNA Kit (NEB, USA).

Techniques: Mutagenesis, Sequencing

Journal: Human Reproduction (Oxford, England)

Article Title: A homozygous variant in HFM1 causes preimplantation embryo developmental arrest by disrupting zygotic genome activation

doi: 10.1093/humrep/deaf238

Figure Lengend Snippet: Minigene assays and pathogenicity prediction to assess the pathogenicity of the homozygous variants in HFM1 . ( A ) Representative images showing the morphology of MII oocytes and embryo development on Days 3 and 4 after IVF from the control and proband II-3, who carries the homozygous HFM1 variant. In two independent ART attempts, all arrested embryos displayed similar morphology. ( B, C ) Prediction results from Mutation Taster and splice site prediction tools. In (B), higher ‘Model’ scores indicate greater prediction reliability. In (C), the ‘-’ symbol denotes the loss of splice sites caused by the HFM1 mutation. ( D ) Overview of sample processing and transcriptomic analysis in human eight-cell embryos. ( E ) RNA-seq analysis of HFM1 transcripts in control and mutant embryos, revealing altered mRNA splicing patterns in HFM1 mutant embryos. ( F ) Schematic diagram of plasmid transfection into HEK293T and HeLa cells, sample collection, mRNA extraction, reverse transcription, and agarose gel electrophoresis. ( G ) Minigene assays and corresponding HFM1 mRNA bands in HEK293T and HeLa cells. Red arrows indicate mRNA bands corresponding to wild-type and mutant constructs. Images are representative of three independent experiments. ( H ) Schematic diagram illustrating aberrant mRNA splicing caused by the HFM1 mutation. The wild-type plasmid produces a single 364 bp mRNA band, whereas the mutant plasmid generates three bands: 447 bp, 266 bp, and 136 bp, corresponding to Intron 24 retention, Exon 24 skipping, and Exons 24 and 25 skipping, respectively.

Article Snippet: The target gene fragments were amplified by PCR, and in vitro transcription of capped mRNA was performed using the HiScribe ® T7 ARCA mRNA Kit (NEB, USA).

Techniques: Control, Variant Assay, Mutagenesis, RNA Sequencing, Plasmid Preparation, Transfection, Extraction, Reverse Transcription, Agarose Gel Electrophoresis, Construct

Journal: bioRxiv

Article Title: The dynamics and strategy of RNA replication in astroviruses

doi: 10.64898/2026.01.19.700307

Figure Lengend Snippet: ( A ) Northern blot analysis of poly(A)-selected RNA isolated from infected Caco-2 cells (500 ng per sample); 100 ng of genomic and 25 ng of subgenomic in vitro transcribed T7 HAstV1 RNA were used as a control. ( B ) Caco-2 cells were infected with HAstV1 at MOI 5 and harvested at 6, 12 or 18 hpi in duplicate. Bar graphs show the density of mapped fragments in the gRNA/sgRNA-overlap region (pink), upstream of the sgRNA region (red), and the difference (yellow). Coverage is quantified as fragments per kilobase per million fragments mapped to vRNA(+) or host mRNA(+). Fragments mapping to the sgRNA region may derive from either gRNA or sgRNA; the difference (yellow) in density between the sgRNA and non-sgRNA regions was used to estimate the relative abundance of sgRNA, whereas the density in the non-sgRNA region (red) was used to estimate the relative abundance of gRNA. (C) Relative densities of (+)gRNA, (+)sgRNA, (−)gRNA and (−)sgRNA. Numbers below bars show the estimated sgRNA:gRNA ratio (1 d.p.). ( D ) Same data as panel (B), plotted on a log scale. ( E ) Estimated (−):(+) ratio for gRNA and sgRNA species. ( F ) Length 6785 nt gRNAs (red), together with sgRNAs (yellow) beginning at nt 4315, in a 1:1 ratio, were randomly fragmented in silico , using a fixed 1/60 probability of cleavage between any adjacent pair of nucleotides. Then fragments in the 50–70 nt length range were selected. The top panel schematically illustrates the first 100 fragments that map at least partly within the region from 120 nt 5ʹ to 120 nt 3ʹ of nt 4315. The middle panel shows a histogram of the 5ʹ end positions of these 100 fragments. The bottom panel shows an equivalent histogram for the first 100,000 similarly selected fragments. ( G ) Caco-2 cells were infected with HAstV1 at MOI 5 and harvested at 18 hpi, with proteinase K (PK) treatment. Histograms show positions of 5ʹ ends of fragments mapping to vRNA(+) (red), 3ʹ ends of fragments mapping to vRNA(−) (blue), and total coverage of vRNA(+) and vRNA(−) (orange and pale blue, respectively). For the 5ʹ/3ʹ-end histograms, counts were normalized to fragments per million fragments mapped to vRNA(+) or host mRNA(+) (’reads per million’; RPM). For the 3ʹ end plot, the histogram shows the 3ʹ ends of negative-sense fragments, corresponding to 5ʹ ends of the positive-sense reverse complements of the fragments. For the total coverage plot, the y -axis scale is arbitrary, but vRNA(−) coverage is scaled relative to vRNA(+) coverage by the indicated factor to aid visualization. The figure is for tech. rep. 1, 18 hpi, PK, biol. rep. 2; see Figs S5-S6 and S8-S11 for 5ʹ/3ʹ-end and total coverage histograms for all 16 samples. (H) As for panel (G) but for HAstV4, 24 hpi, biol. rep. 1; see Figs S13-21 for the full set of histograms for HAstV4, MLB1, MLB2 and VA1 infections.

Article Snippet: Linearized replicon-encoding plasmids were used to generate T7 RNAs using HighYield T7 ARCA mRNA Synthesis Kit (Jena Bioscience, RNT-102) according to the manufacturer’s instructions, purified using Zymo RNA Clean & Concentrator kit, and quantified.

Techniques: Northern Blot, Isolation, Infection, In Vitro, Control, In Silico