mRNAs were isolated from the testes of WT and repro57 mutant mice at 10 weeks of age using TRIzol (Invitrogen, CA, USA) following the manufacturer’s instructions. Genomic DNA in the RNA sample was digested using DNaseI (Takara Bio Inc., Shiga, Japan), and the RNA solutions were purified by phenol/chloroform treatment. cDNAs were synthesized by reverse transcription reaction using Superscripts III reverse transcriptase (Invitrogen) and Oligo dT primer (Invitrogen). RNAs in the cDNA solution were then digested with RNase H (Toyobo, Osaka, Japan). Given evidence for splice variants of Rnf212 (Reynolds et al. 2013 (link)) (http://www.ncbi.nlm.nih.gov/nuccore/XM_006535331.1 , http://www.ncbi.nlm.nih.gov/nuccore/XM_006535332.1 , http://www.ncbi.nlm.nih.gov/nuccore/XM_001476621.5 ), the sequences of the variant-specific primers, how they align to the exonic structure, and the PCR cycles are shown in Supplementary Figure 1 .
Rnase h
Manufactured by Toyobo
Sourced in Japan
RNase H is a laboratory enzyme that specifically degrades the RNA strand in RNA-DNA hybrid molecules. It plays a crucial role in various molecular biology techniques by removing the RNA template during DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Takara Bio (1 mentions)
Superscripts 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rq rnase free dnase, by Promega (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
2 protocols using rnase h
1
Characterization of Rnf212 Splice Variants
2
Quantitative RT-PCR Gene Expression Analysis
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Total cellular RNA was isolated using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer. The samples were treated with RQ-RNase-free DNase (Promega, Madison, WI, USA). An RNaseH (Toyobo, https://www.toyobo-global.com/ ) reverse transcription kit was used to synthesize cDNA, according to the manufacturer’s instructions (Promega). qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Amplification and quantification were performed with gene-specific primers using CFX Manager software (Bio-Rad), and values were normalized against internal UBIQ1. Three biological and two technical replicates were used for each analysis [46 (link)]. All the primers used for qRT-PCR are listed in Table S1 .
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