high yield t7 arca mrna synthesis kit  (Jena Bioscience)

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

Journal: Nucleic Acids Research

Article Title: Small nucleolar RNAs promote the restoration of muscle differentiation defects in cells from myotonic dystrophy type 1

doi: 10.1093/nar/gkaf232

Figure Lengend Snippet: Functional studies of SNORA40 and SNORA70 in muscle cells. Immunofluorescence in normal MT with (ASO CTRL) or without (ASOs SNORA40 and SNORA70) the presence of ( A ) SNORA40 and ( E ) SNORA70 ( n ≥ 3). Relative fusion index measured at 5 days post-differentiation after the loss of ( B ) SNORA40 and ( F ) SNORA70 in normal muscle cells. More than 800 nuclei per replicate were counted to calculate the fusion index ( n ≥ 3). Relative expression of the CCND3 and MYOG mRNA differentiation markers in normal muscle cells depleted in ( C ) SNORA40 and ( G ) SNORA70 ( n ≥ 3). Relative expression of the MYF5 and CCNB1 mRNA proliferation markers in normal muscle cells depleted in ( D ) SNORA40 and ( H ) SNORA70 ( n ≥ 3). Immunofluorescence in DM1 MT with plasmids expressing either a random sequence (Rd), ( I ) SNORA40 (SNORA40), or ( M ) SNORA70 ( n ≥ 3). Relative to the fusion index measured at 5 days post-differentiation after the gain of ( J ) SNORA40 and ( N ) SNORA70 in DM1 muscle cells. More than 800 nuclei per replicate were counted to calculate the fusion index ( n ≥ 3). Relative expression of the CCND3 and MYOG mRNA differentiation markers in DM1 muscle cells expressing ( K ) SNORA40 and ( O ) SNORA70 ( n ≥ 3). Relative expression of the MYF5 and CCNB1 mRNA proliferation markers in DM1 muscle cells expressing ( L ) SNORA40 and ( P ) SNORA70 ( n ≥ 3). ASO, gapmers; CTRL, control; Alpha-tub, alpha-tubulin; FI, fusion index; U6, RNU6; CCND3, Cyclin D3; MYOG, Myogenin; MYF5, Myogenic factor 5; CCNB1, Cyclin B1; d, day of differentiation. Scale bar = 10 μm. Error bars represent the standard error of the mean (SEM). * indicates P -value< .05. Significant differences were assessed by comparing the two conditions: ASO CTRL versus ASO SNORA40 at each corresponding time point. Statistical analyses were performed using a one-way ANOVA Fisher-means comparison test for the Fl results and a two-way ANOVA Tukey’s multiple comparisons test for the qPCR results.

Article Snippet: Full-length CCND3 ivt + Ψ was in vitro transcribed using the HighYield T7 mRNA Synthesis Kit (Ψ-UTP, Jena Bioscience, Germany) before IP.

Techniques: Functional Assay, Immunofluorescence, Expressing, Sequencing, Control, Comparison

Journal: Nucleic Acids Research

Article Title: Small nucleolar RNAs promote the restoration of muscle differentiation defects in cells from myotonic dystrophy type 1

doi: 10.1093/nar/gkaf232

Figure Lengend Snippet: Interaction of CCND3 with SNORA70 and SNORA40. ( A and B ) Physical interaction of CCND3 with SNORA40 and SNORA70, respectively, as predicted with snoGPS software. Scores are indicated in bold. ( C ) The accumulation of CCND3 in the nucleoli. Isolation of nucleoli by sucrose cushion centrifugation was performed as described in . The total RNA from each fraction was extracted and the localization of CCND3 mRNA was assessed by RT-PCR ( n = 2). To confirm the correct isolation of the nucleoli, MALAT1 RNA and SNORD16 were used as nuclear (negative control) and nucleolar (positive control) markers, respectively. cDNA (50 ng of equivalent RNA) was used for each PCR reaction. ( D and E ) Direct interaction of CCND3 with SNORA40 and SNORA70, respectively. U6 and ACA36B were used as negative controls ( n = 2). ( F ) Immunoprecipitation (1 μg of RNA) of Psi in mRNA-enriched populations followed by RT-PCR. Five percent of the input was used. In vitro transcribed CCND3 RNA with and without pseudouridines were used as positive and negative controls, respectively ( n = 3). Ψ, Psi; T, total fraction; C, cytoplasmic fraction; N, nuclear fraction; Np, nucleoplasmic fraction; Nuc, nucleolar fraction; Blk, mock PCR; RNA PD, RNA pull-down; IP, Immunoprecipitation; Ig, immunoglobulin; 18S, rRNA 18S; SH3BGRL3, SH3 domain binding glutamic acid-rich-like protein 3.

Article Snippet: Full-length CCND3 ivt + Ψ was in vitro transcribed using the HighYield T7 mRNA Synthesis Kit (Ψ-UTP, Jena Bioscience, Germany) before IP.

Techniques: Software, Isolation, Centrifugation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Immunoprecipitation, In Vitro, Binding Assay