Journal: bioRxiv

Article Title: Identification of a microRNA with a mutation in the loop structure in the silkworm Bombyx mori

doi: 10.64898/2026.03.24.714027

Figure Lengend Snippet: ( a ) Positional cloning of chromosome 23. Each number indicates the chromosome position of the marker listed in S2 Table. The op locus was located within a region approximately 0.32 Mb in length between 9,598,832 and 9,916,751 bp on chromosome 23. ( b ) Predicted genes within the narrowed region. This region contains four predicted genes, namely, KWMTBOMO13806 , KWMTBOMO13807 , KWMTBOMO13808 , and KWMTBOMO13809 , and one miRNA, namely, bmo-mir-3260 . ( c ) Prediction of the miRNA secondary structure. The secondary structure was predicted on the basis of the miR-3260 nucleotide sequence via RNAfold WebServer. Wild-type indicates wild-type mir-3260 , and mutant indicates m mir-3260 . The black box indicates the mature sequence region, and the red box indicates missing nucleotide sequences in the miR-3260 mutant.

Article Snippet: TaqManTM Fast Advanced Master Mix (Thermo Fisher, Waltham, Massachusetts, USA) and the miR-3260 primers (Thermo Fisher, TaqMan miRNA Assay, Assay ID: 241990_mat) were mixed with each miRNA cDNA library, and quantitative RT‒PCR was performed using the Step One Plus Real‒Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Cloning, Marker, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Identification of a microRNA with a mutation in the loop structure in the silkworm Bombyx mori

doi: 10.64898/2026.03.24.714027

Figure Lengend Snippet: ( a ) miR-3260 expression during development. Embryo 120 h and 216 h indicate 120 and 216 h after oviposition, respectively; 1L, 2L, and 3L indicate first-, second-, and third-instar larvae, respectively; 4L-day 0 and 4L-day 2 indicate days 0 day and 2 of the fourth-instar larvae, respectively; 5L-day 0 and 5L-day 2 indicate days 0 day and 2 of the fifth-instar larvae, respectively. Whole-body miRNA expression levels are presented as RQ values, representing relative expression levels normalized against first-instar larvae samples (RQ = 1). Error bars represent RQ minimum and maximum levels based on standard deviation. U6 was used as an endogenous control. All sample sets were assayed in triplicate as technical replicates. ( b ) miR-3260 expression in the corpora allata. The miRNA expression levels in the corpora allata are presented as RQ values, which represent the relative expression levels calculated by assuming the value for whole bodies of 4-day fifth-instar larval samples as 1. Error bars indicate RQ minimum and maximum levels based on standard deviation. U6 was used as an endogenous control. All sample sets were assayed in triplicate as technical replicates.

Article Snippet: TaqManTM Fast Advanced Master Mix (Thermo Fisher, Waltham, Massachusetts, USA) and the miR-3260 primers (Thermo Fisher, TaqMan miRNA Assay, Assay ID: 241990_mat) were mixed with each miRNA cDNA library, and quantitative RT‒PCR was performed using the Step One Plus Real‒Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Expressing, Standard Deviation, Control

Journal: bioRxiv

Article Title: Identification of a microRNA with a mutation in the loop structure in the silkworm Bombyx mori

doi: 10.64898/2026.03.24.714027

Figure Lengend Snippet: mRNA sequences that bind to miR-3260 were predicted using RNAhybrid. The minimum free energy value (mfe), which indicates the ease of binding, was set to −20 or less, and the seed sequence was allowed to have a G:U wobble.

Article Snippet: TaqManTM Fast Advanced Master Mix (Thermo Fisher, Waltham, Massachusetts, USA) and the miR-3260 primers (Thermo Fisher, TaqMan miRNA Assay, Assay ID: 241990_mat) were mixed with each miRNA cDNA library, and quantitative RT‒PCR was performed using the Step One Plus Real‒Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Identification of a microRNA with a mutation in the loop structure in the silkworm Bombyx mori

doi: 10.64898/2026.03.24.714027

Figure Lengend Snippet: ( a ) The upper graph shows the hatching rate; the lower panel shows the phenotype of the first-instar larva. ns indicates non significance. ( b ) Jhamt mRNA expression after injection of the miR-3260 mimic or inhibitor. ( c ) KWMTBOMO13816 mRNA expression after injection of the miR-3260 mimic or inhibitor; mRNA expression levels in each sample are presented as RQ values, which represent the relative expression levels calculated assuming the value for the control samples as 1. Error bars represent RQ minimum and maximum levels based on standard deviation. Rp49 was used as an endogenous control.

Article Snippet: TaqManTM Fast Advanced Master Mix (Thermo Fisher, Waltham, Massachusetts, USA) and the miR-3260 primers (Thermo Fisher, TaqMan miRNA Assay, Assay ID: 241990_mat) were mixed with each miRNA cDNA library, and quantitative RT‒PCR was performed using the Step One Plus Real‒Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Expressing, Injection, Control, Standard Deviation

Journal: bioRxiv

Article Title: Identification of a microRNA with a mutation in the loop structure in the silkworm Bombyx mori

doi: 10.64898/2026.03.24.714027

Figure Lengend Snippet: A dicing assay was performed using crude extracts prepared from BmN cells as enzyme fractions and 32 P-end-labeled ssRNA or dsRNA as substrates. The cleaved RNAs were detected as band intensities with a Typhoon FLA 7000 image analyzer. Schematic diagram of the dicing assay used in this study: ( a ) 50-nt dsRNA, ( b ) wild-type bmo-let7 precursor, and ( c ) wild-type miR-3260 precursor. The red star indicates 32 P. ( d ) The 50-nt dsRNA was used as a dicing assay experimental control. The bmo-let7 precursor ( pre-bmo-let7 ) was used as a dicing assay positive control. The mutant bmo-let7 precursor ( pre-bmo- m let7 ) was used as a dicing assay negative control. Wild-type and mutant of the miR-3260 precursor. Lane M, RNA size markers of 50, 24, and 21 nt; NC, negative control (no-enzyme fraction); FB, fat body; CC, cultured BmN cells. Red or gray asterisks indicate diced products by BmDicer-1 and BmDicer-2, respectively.

Article Snippet: TaqManTM Fast Advanced Master Mix (Thermo Fisher, Waltham, Massachusetts, USA) and the miR-3260 primers (Thermo Fisher, TaqMan miRNA Assay, Assay ID: 241990_mat) were mixed with each miRNA cDNA library, and quantitative RT‒PCR was performed using the Step One Plus Real‒Time PCR system (Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Labeling, Control, Positive Control, Mutagenesis, Negative Control, Cell Culture