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 .
Superscripts 3 reverse transcriptase
Manufactured by Thermo Fisher Scientific
Superscript III reverse transcriptase is a thermostable reverse transcriptase enzyme used for the conversion of RNA to complementary DNA (cDNA). It provides efficient and sensitive reverse transcription of RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Takara Bio (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Toyobo (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
2 protocols using superscripts 3 reverse transcriptase
1
Characterization of Rnf212 Splice Variants
2
Quantitative Real-Time PCR Analysis of Rat Ovarian Gene Expression
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The ovarian tissues were homogenized with liquid nitrogen. Total RNA was isolated from individual rat ovarian tissues using TRIzol reagent (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The total RNA concentration was measured by means of a Nanodrop spectrophotometer (Thermo Fisher Scientific) and reverse transcribed into cDNA using Superscripts III reverse transcriptase (Invitrogen). The PCR conditions for the synthesis of cDNA were as follows: 5 min at 65 °C, 1 min at 4 °C, 60 min at 50 °C, and 15 min at 72 °C. The cDNA was used for qRT–PCR analysis with SYBR Ex Taq (Roche, Basel, Switzerland). The cDNA was amplified by PCR under the following conditions: 5 s at 95 °C, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The sequences of the qRT–PCR primers are listed in Table 2 . rGAPDH was used as an internal control for normalization, and each sample was analyzed in triplicate.
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