hiscribe t7 arca mrna kit  (New England Biolabs)

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