reverse transcription kit  (Vazyme Biotech Co)

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: lncRNA TDRG1 acts as a piRNA precursor to generate piRNAs A. RT‒qPCR analysis of TDRG1 overexpression in GC-2 spd(ts) cells. Gapdh was used as the internal reference; empty vector transfection as the control (Ctrl). The relative expression was calculated relative to the expression level of Gapdh . Relative expression was normalized to Gapdh , and calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗∗ P < 0.001). B. Expression levels of TDRG1 -related piRNAs after TDRG1 overexpression in GC-2 spd(ts), detected by RT‒qPCR. U6 snRNA was used as the internal reference; empty vector transfection as the control (Ctrl). Three piRNAs (piR-128687, piR-115293 and piR-103374) with no sequences overlap to 1700008K24Rik were used as negative controls. Relative expression was normalized to U6 , and calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS: no significant difference).

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: Over Expression, Plasmid Preparation, Transfection, Control, Expressing

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: RNA-seq analysis of 1700008K24Rik -overexpressing GC-2 spd(ts) cells A . Volcano plot displaying differentially expressed genes (DEGs) in GC-2 spd(ts) cells overexpressing 1700008K24Rik versus control, based on RNA-seq data. The x-axis shows log 2 (fold change) the y-axis shows -log 10 ( P value). Red, green, and gray dots indicate up-regulated, down-regulated and non-DEGs, respectively. B-C . Gene Ontology (GO) enrichment analysis of up-regulated (B) and down-regulated (C) genes after 1700008K24Rik overexpression in GC-2 spd(ts) cells. Selected biological processes that were potentially involved are listed on the right. D . KEGG pathway enrichment analysis of down-regulated genes after 1700008K24Rik overexpression in GC-2 spd(ts) cells. Related pathways that were potentially involved are listed on the right. E-F . RT‒qPCR validation of RNA-seq results for up-regulated (E) and down-regulated (F) genes following 1700008K24Rik overexpression in GC-2 spd(ts) cells. Gapdh was used as the internal control; empty vector transfectionas the experimental control (Ctrl). Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗ P < 0.05, ∗∗ P < 0.01, NS: not significant). G . Predicted targets of the 1700008K24Rik-related piRNAs among the differentially expressed genes, analyzed using miRanda algorithm. Shown are eight spermatogenesis-related genes harboring potential piRNA targeting sites.

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: RNA Sequencing, Control, Over Expression, Biomarker Discovery, Plasmid Preparation, Expressing

Journal: Non-coding RNA Research

Article Title: The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor

doi: 10.1016/j.ncrna.2026.04.001

Figure Lengend Snippet: Knockdown of 1700008K24Rik impairs mouse spermatogenesis. A . Visualization of AAV9 virus delivery into mouse seminiferous tubules, with trypan blue indicating successful injection. B . RT‒qPCR analysis of 1700008K24Rik knockdown efficiency in mouse testes. Three-week-old mice were injected with AAV9-shRNA-GFP (shRNA) and analyzed after one month. The contralateral testis injected nontargeted shRNA (shCtrl) served as the control. Gapdh was used as the internal reference. Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗∗ P < 0.01, NS: not significant). C . Representative images of testis size AAV9-shRNA-GFP (shRNA) and AAV9-shCtrl-GFP (shCtrl) injected mice. Viruses were delivered at 3 weeks of age; tissues were collected one month post-injection. n = 3. D . Testis weight quantification of shRNA and shCtrl groups. Relative testis weight was normalized to shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗∗ P < 0.001), n = 3. E . H&E staining of paraffin-embedded testis sections from shRNA and shCtrl groups. Scale bars: 100 μm (overview) and 20 μm (magnified). F . Seminiferous epithelium thickness measurement in shRNA and shCtrl groups. Thickness was assessed from five H&E-stained sections per testis. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗ P < 0.05, ∗∗ P < 0.01). G . Epididymal sperm counts in shRNA and shCtrl groups. Relative sperm count was calculated versus shCtrl. Data are shown as mean ± SD; n = 3. Statistical significance was determined by unpaired t -test (∗∗ P < 0.01), n = 3. H . Expression levels of 1700008K24Rik -related piRNAs in shRNA or shCtrl testes, detected by RT‒qPCR. Three piRNAs (piR-128687, piR-115293 and piR-103374) with no sequence overlap to 1700008K24Rik were used as negative controls. Relative expression was calculated versus Ctrl. Data are presented as the mean ± SD; n = 3. Statistical significance was determined by unpaired t- test (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS: not significant).

Article Snippet: Reverse transcription of mRNAs or lncRNAs was performed using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme, Cat# R212-01), while reverse transcription of piRNAs was carried out using a piRNA reverse transcription kit (miRNA First Strand cDNA Synthesis Kit (Tailing Reaction), Sangon Biotech, Cat# B532451). qPCR was performed via qPCR reagent (AceQ qPCR SYBR Green Master Mix, Vazyme, Cat# Q111-02) on a qPCR detection system (Bio-Rad) using the specific primers listed in in the “Multimedia component 3” file. mRNA or lncRNA expression was calculated, referring to that of the gene Gapdh , while piRNA expression was calculated, referring to that of the gene U6 .

Techniques: Knockdown, Virus, Injection, shRNA, Control, Expressing, Staining, Sequencing