nde i restriction enzyme  (New England Biolabs)

Journal: bioRxiv

Article Title: Investigating dietary microRNA stability and function using a transgenic milk model with unique microRNA sequences

doi: 10.64898/2026.04.13.718217

Figure Lengend Snippet: The above plasmid is a mammalian expression plasmid carrying the open reading frames coding for the following three genes: mCherry (a fluorescent protein whose signal was used to assess transfection efficiency between different conditions upon plasmid transfection into HEK293T cells), ampicillin resistance gene (AmpR, the antibiotic resistance gene used for bacterial selection during plasmid cloning) and the gene coding for β-lactoglobulin (cwPAEP). NdeI restriction sites are denoted on the map (NdeI highlighted in green). The vector was custom-designed by VectorBuilder Inc. The sequence of the plasmid was obtained from the VectorBuilder Inc. database, and the map was built using the VectorBee software 2.5.0-WIN.

Article Snippet: The same was done for the absolute quantification of β-lactoglobulin but the starting material for establishing the standard curve was β-lactoglobulin-coding sequence (represented by cPAEP segment on plasmid map) obtained from β-lactoglobulin plasmid digestion with NdeI (New England Biolabs, Ipswich, MA, USA, Cat. No. R0111S) restriction enzyme (see on plasmid map, ).

Techniques: Plasmid Preparation, Expressing, Transfection, Selection, Cloning, Sequencing, Software

Journal: bioRxiv

Article Title: Investigating dietary microRNA stability and function using a transgenic milk model with unique microRNA sequences

doi: 10.64898/2026.04.13.718217

Figure Lengend Snippet: A. Detection of β-lactoglobulin protein in WT (WT cow #1603 lot) and TG (TG #1508 lot 1 and lot 2) non-fractioned skim milk samples. B. Small RNA sequencing of TG (TG #1507) and WT (TG # 1603) milk. Presented are the reads per million (RPM) of the four AmiRs (AmiR-4*, AmiR-4, AmiR-3*, and AmiR-3) along with 11 other classical milk microRNAs. These classical milk microRNAs are commonly known to be highly enriched in milk of different types and from different species.

Article Snippet: The same was done for the absolute quantification of β-lactoglobulin but the starting material for establishing the standard curve was β-lactoglobulin-coding sequence (represented by cPAEP segment on plasmid map) obtained from β-lactoglobulin plasmid digestion with NdeI (New England Biolabs, Ipswich, MA, USA, Cat. No. R0111S) restriction enzyme (see on plasmid map, ).

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: Investigating dietary microRNA stability and function using a transgenic milk model with unique microRNA sequences

doi: 10.64898/2026.04.13.718217

Figure Lengend Snippet: HEK293T cells were co-transfected with a plasmid expressing β-lactoglobulin along with different concentrations of AmiR-3/4 mimics. A. Scheme depicting the binding sites of AmiR-3 and AmiR-4 in the coding sequence of β-lactoglobulin mRNA (Genbank no. X 14712.1). B-C Western blots, densitometry, and RT-qPCR-based absolute quantification of β-lactoglobulin protein and mRNA levels in HEK293T cells after co-transfection with β-lactoglobulin plasmid and AmiR-3 mimic at three different concentrations (10 pM, 50 pM, and 100 pM). D-E Western blots, densitometry, and RT-qPCR-based absolute quantification of β-lactoglobulin protein and mRNA levels in HEK293T cells after co-transfection with β-lactoglobulin plasmid and AmiR-4 mimic at three different concentrations (10 pM, 50 pM, and 100 pM). F. Western blots and densitometry quantification of β-lactoglobulin protein levels in HEK293T cells after co-transfection with β-lactoglobulin plasmid, AmiR-3, and AmiR-4 (simultaneously or individually). The microRNA mimic mock, Cel-67, was used as a negative control at 100 pM or 5 nM. Proteins and total RNA were extracted from cells and used for analysis 24 h following mimic transfection. GAPDH was used as an internal control for western blot densitometry analysis and for RT-qPCR. The values are means ± SD of three independent experiments (n=3-4). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). G. Table showing the equivalent copy number of AmiRs required to inhibit β-lactoglobulin protein and/or mRNA expression under specific experimental conditions (transfection of 38,000 HEK293T cells with 50ng β-lactoglobulin-expressing plasmid). CDS = Coding sequence.

Article Snippet: The same was done for the absolute quantification of β-lactoglobulin but the starting material for establishing the standard curve was β-lactoglobulin-coding sequence (represented by cPAEP segment on plasmid map) obtained from β-lactoglobulin plasmid digestion with NdeI (New England Biolabs, Ipswich, MA, USA, Cat. No. R0111S) restriction enzyme (see on plasmid map, ).

Techniques: Transfection, Plasmid Preparation, Expressing, Binding Assay, Sequencing, Western Blot, Quantitative RT-PCR, Quantitative Proteomics, Cotransfection, Negative Control, Control

Journal: bioRxiv

Article Title: Investigating dietary microRNA stability and function using a transgenic milk model with unique microRNA sequences

doi: 10.64898/2026.04.13.718217

Figure Lengend Snippet: Scheme depicting the binding sites of AmiR-3 and AmiR-4 in the coding sequence of β-lactoglobulin mRNA (Genbank no. X 14712.1).

Article Snippet: The same was done for the absolute quantification of β-lactoglobulin but the starting material for establishing the standard curve was β-lactoglobulin-coding sequence (represented by cPAEP segment on plasmid map) obtained from β-lactoglobulin plasmid digestion with NdeI (New England Biolabs, Ipswich, MA, USA, Cat. No. R0111S) restriction enzyme (see on plasmid map, ).

Techniques: Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Investigating dietary microRNA stability and function using a transgenic milk model with unique microRNA sequences

doi: 10.64898/2026.04.13.718217

Figure Lengend Snippet: HEK293T cells were co-transfected with a plasmid expressing β-lactoglobulin along with different concentrations of AmiR-3/4 mimics. A-B. Western blots, densitometry, and RT-qPCR-based absolute quantification of β-lactoglobulin protein and mRNA levels in HEK293T cells after co-transfection with β-lactoglobulin plasmid and AmiR-3 mimic at three different concentrations (10 nM, 20 nM, and 30 nM) for 24 h (n=3). C-D. Western blots, densitometry, and RT-qPCR-based absolute quantification of β-lactoglobulin protein and mRNA levels in HEK293T cells after co-transfection with β-lactoglobulin plasmid and AmiR-3 mimic at three different concentrations (10 nM, 20 nM, and 30 nM) for 48 h (n=2). The microRNA mimic mock, Cel-67, was used as a negative control at 30 nM. GAPDH was used as an internal control for western blot densitometry analysis and for RT-qPCR. The values are means ± SD of three independent experiments (n=2-3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: The same was done for the absolute quantification of β-lactoglobulin but the starting material for establishing the standard curve was β-lactoglobulin-coding sequence (represented by cPAEP segment on plasmid map) obtained from β-lactoglobulin plasmid digestion with NdeI (New England Biolabs, Ipswich, MA, USA, Cat. No. R0111S) restriction enzyme (see on plasmid map, ).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR, Quantitative Proteomics, Cotransfection, Negative Control, Control