biotin rna labeling mix  (New England Biolabs)

Journal: Epigenetics

Article Title: A novel lncRNA, Lnc21q22.11, suppresses gastric cancer growth by inhibiting MEK/ERK pathway.

doi: 10.1080/15592294.2025.2512764

Figure Lengend Snippet: Figure 1. Isolation and identification of Lnc21q22.11 in gastric cancer. (a) RNA-seq data showing the Fold changes in lncRNA expression levels in gastric cancer compared to adjacent tissue samples. (b) Real-time PCR results showing reduced expression of Lnc21q22.11 in cancer tissue compared to adjacent tissue samples (n = 245). ***p < 0.001. (c) RT-PCR results showing the expression of Lnc21q22.11 in gastric cancer cell lines. H2O: negative control; GAPDH: internal control. (d) Northern blotting showing the size of Lnc21q22.11, 1000 nt and 1500 nt were marker sizes. GAPDH served as an RNA expressing control. (e) 5'-RACE and 3'-RACE results obtained after walking-PCR extension. (f) Three potential coding patterns of the full length Lnc21q22.11 sequence were constructed into pcDNA3.1 expression vectors. ZSCAN23-Flag fusion protein served as a positive control. FL: full length sequence. (g) Different patterns of Lnc21q22.11 constructs were transfected into AGS cells and detected with Flag antibody. The size of ZSCAN23-Flag was 47 kDa. β-actin: internal control. (h) Cytoplasmic and nuclear RNA were separated from BGC-823 cells, and Lnc21q22.11 was detected by real-time PCR. U6: control for nucleus; GAPDH: control for cytoplasm. (i) FISH assay showing localization of Lnc21q22.11 in BGC- 823 cells. The scale bar represents 25 μm. (j) Histone modification status of the Lnc21q22.11 promoter region predicted by cistrome DB database. H3K4me3 enrichment locations indicate transcriptional activation sites in AGS, BGC-823, and MGC-803 cells. (k) Histone modification status in Lnc21q22.11 promoter region detected by ChIP-PCR assay with H3K4me3, H3K9me2, H3K27me3 antibodies.

Article Snippet: Northern blotting, 5'- and 3'- rapid amplification of cDNA ends Biotin-labeled Lnc21q22.11 probes were synthesized with T7 RNA polymerase using a biotin RNA labeling mix (NEB, USA, #M0251).

Techniques: Isolation, RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Control, Northern Blot, Marker, Sequencing, Construct, Positive Control, Transfection, Modification, Activation Assay

Journal: Epigenetics

Article Title: A novel lncRNA, Lnc21q22.11, suppresses gastric cancer growth by inhibiting MEK/ERK pathway.

doi: 10.1080/15592294.2025.2512764

Figure Lengend Snippet: Figure 4. Interaction of Lnc21q22.11 and MYH9, and their co-localization in the nucleus. (a) RNA pull-down assay showing Lnc21q22.11 and protein complexes. The red arrow indicates the differential band. LacZ probes were used for the control group. (b) Predicted interaction between Lnc21q22.11 and MYH9 using Alphafold3. Lnc21q22.11 and MYH9 are represented by ribbon and surface, respectively. Short red bands indicate hydrogen bonds between Lnc21q22.11 and MYH9. (c) Interaction between Lnc21q22.11 and MYH9 verified by RNA pull-down and western blotting using labeled Lnc21q22.11 probes and MYH9 antibodies. (d) Interaction between Lnc21q22.11 and MYH9 validated by RNA immunoprecipitation assay. IgG antibody was used for the control group. (e) Confocal laser microscopic images showing the localization of Lnc21q22.11 (red) and MYH9 (green) in cells, and their co- localization in the nucleus. The scale bar represents 25 μm.

Article Snippet: Northern blotting, 5'- and 3'- rapid amplification of cDNA ends Biotin-labeled Lnc21q22.11 probes were synthesized with T7 RNA polymerase using a biotin RNA labeling mix (NEB, USA, #M0251).

Techniques: Pull Down Assay, Control, Western Blot, Labeling, RNA Immunoprecipitation

Journal: EMBO Reports

Article Title: PIF/harbinger transposon-derived protein promotes 7SL expression to enhance pathogen resistance

doi: 10.1038/s44319-025-00379-8

Figure Lengend Snippet: ( A ) RNA pull-down analysis for cell lysates (input), unlabeled 7SL probe (negative control) pull down, biotin-labeled 7SL probe pull down (experimental group) and NARL probe pull down (positive control). ( B ) The Ago2-RIP assay for the amount of 7SL in MKCs transfected Ago2-Flag or pcDNA3.1-Flag. ** P < 0.0001, values compared to IgG group by Student’s t test. ( C ) A schematic illustration showing overlapping of the target miRNAs of 7SL predicted by TargetScan and miRanda. ( D ) Relative expression of candidate miRNAs in MKCs transfected with 7SL plasmids or si-7SL. ** P < 0.01, values compared to control group by Student’s t test. 7SL: P = 0.0006. si-7SL: P = 0.0002. ( E ) The luciferase activity of constructed miR-2187-3p sensor in MKC cells transfected with indicated mimics and plasmids. ** P < 0.01, values compared to control group by Student’s t test. miR-2187-3p: P = 0.0001. 7SL: P = 0.0001. ( F ) The luciferase activity of Luc-7SL in MKC cells transfected with miR-2187-3p. miR-2187-3p-binding sites in 7SL wild-type form (Luc-7SL-wt) and the mutated form (Luc-7SL-mut) were shown. ** P = 0.0002, values compared to control group by Student’s t test. ( G ) MS2-RIP method was used to identify the binding between 7SL-wt or 7SL-mut and miR-2187-3p in MKC cells. ** P = 0.0003, values compared to MS2 group by Student’s t test. ( H ) RNA pull-down assay to detect the binding between 7SL-wt or 7SL-mut and miR-2187-3p. ** P < 0.0001, values compared to control group by Student’s t test. ( I ) The luciferase activity of TRAF6 3′UTR in MKC cells transfected with miR-2187-3p. miR-2187-3p binding sites in wild-type of TRAF6 3′UTR (TRAF6-3′UTR-wt) and a mutated form of 3′UTR (TRAF6-3′UTR-mut) were shown. ** P = 0.0002, values compared to control grou p by Student’s t test. ( J , K ) The expression of TRAF6 in MKC cells transfected with indicated mimics was determined by qPCR ( J ) and Western blotting ( K ). ** P < 0.01, values compared to control group by Student’s t test. miR-2187-3p, P = 0.0003. miR-2187-3p, P = 0.0002. ( L ) mRNA and protein levels of TRAF6 in MKC cells transfected with miR-2187-3p or miR-2187-3p + 7SL plasmid. ** P = 0.0005, values compared to NC group by Student’s t test. ( M ) Luciferase activity of TRAF6-3′UTR in MKC cells transfected with indicated mimics and plasmids. ** P < 0.01, values compared to control group by Student’s t test. miR-2187-3p, P = 0.0001. 7SL, P < 0.0001. ( N ) qPCR analysis of TRAF6 in MKC cells transfected with si-7SL. ** P = 0.0003, values compared to si-Ctrl group by Student’s t test. ( O ) Luciferase activity of NF-κB and IRF3 report genes in MKC cells co-transfected with indicated mimics and plasmids. ** P < 0.01, values compared to control group by Student’s t test. NF-κB: miR-2187-3p, P = 0.0003. 7SL, P = 0.0001. IRF3: miR-2187-3p, P = 0.0061. 7SL, P = 0.0004. ( P , Q ) qPCR analysis of miR - 2187-3p in MKC cells transfected with si-ADF-IL ( P ) or with 7SL and si-ADF-IL( Q ). ** P < 0.01, * P < 0.05,values compared to control group by Student’s t test. si-1, P = 0.0009. si-2, P = 0.0137. 7SL, P = 0.0009. ( R , S ) qPCR analysis of TRAF6 in MKC cells transfected with si - ADF-IL (R) or with 7SL and si-ADF-IL ( S). ** P < 0.01, * P < 0.05, values compared to control group by Student’s t test. si-1, P = 0.0004. si-2, P = 0.0042. 7SL, P = 0.0020. All results are represented as the means ± SE of n = 3 biological replicates. .

Article Snippet: To detect the binding site between 7SL and miR-2187-3p, 7SL and 7SL-mut with miR-2187-3p binding sites mutated were biotin-labeled with the T7 RNA polymerase and Biotin RNA Labeling Mix (RiboBio), treated with RNase-free DNase I, and purified with RNeasy Mini Kit (Qiagen).

Techniques: Negative Control, Labeling, Positive Control, Transfection, Expressing, Control, Luciferase, Activity Assay, Construct, Binding Assay, Pull Down Assay, Western Blot, Plasmid Preparation

Journal: EMBO Reports

Article Title: PIF/harbinger transposon-derived protein promotes 7SL expression to enhance pathogen resistance

doi: 10.1038/s44319-025-00379-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: To detect the binding site between 7SL and miR-2187-3p, 7SL and 7SL-mut with miR-2187-3p binding sites mutated were biotin-labeled with the T7 RNA polymerase and Biotin RNA Labeling Mix (RiboBio), treated with RNase-free DNase I, and purified with RNeasy Mini Kit (Qiagen).

Techniques: Recombinant, Sequencing, Labeling, Immunoprecipitation, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Reporter Assay, Mutagenesis, Software