For 5’-RACE analysis, we used one mouse from CRISPR-Cas9-treated and control groups. The total RNA was isolated from frozen muscle samples and reverse transcribed by Mint revertase (Evrogen, SK001) with the specific primer to Dmd cDNA, Dp71-fl-Rev. Step-out amplification of upstream Dmd sequences was performed with the Mint RACE primer set (Evrogen, SK004) and reverse primers designed for exons 64–66 (Dp71-5′ rev1-3; Table S3 ). The obtained bulk PCR products were purified from gel and ligated into the pAL-TA vector (Evrogen, TA002) with further transformation of ligation mixes into XL1-Blue cells (Evrogen, CC001). Ten clones for each group were sent for Sanger sequencing.
Mint revertase
Manufactured by Evrogen
Mint revertase is a recombinant enzyme used for the reversal of DNA methylation. It catalyzes the removal of 5-methylcytosine from DNA.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 96 sw 1, by Roche (2 mentions)
Aurum total rna mini kit, by Bio-Rad (2 mentions)
Sybr green 1 fluorescent dye, by Evrogen (2 mentions)
Mint race primer set, by Evrogen (1 mentions)
Xl1 blue cells, by Evrogen (1 mentions)
Pal ta vector, by Evrogen (1 mentions)
Mmlv revertase, by Evrogen (1 mentions)
Light cycler 96 real time detection thermal cycler, by Roche (1 mentions)
4 protocols using mint revertase
1
RNA analysis of Duchenne muscular dystrophy
2
Isolation and Characterization of SaNPF6.3 in Suaeda
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Total RNA from S.altissima plant organs was isolated by the hot phenolic method [52 (link)] and used as a template for the total first-strand cDNA synthesis. For amplification of the 3′- and 5′- ends of the SaNPF6.3 transcript by the Step-Out RACE method, the first-strand cDNA was synthesized on the total RNA template, isolated from Suaeda roots, using MINT revertase (Evrogen, Moscow, Russia). Full-length cDNA of SaNPF6.3 gene was also amplified on the total RNA template, isolated from Suaeda roots. To obtain full-length cDNA of SaNPF6.3 and quantify the representation of the gene transcripts in S. altissima organs, first-strand cDNA synthesis was performed on total RNA templates using (dT)15 primer and MMLV revertase (Evrogen, Moscow, Russia).
3
Quantification of Gene Expression using Real-Time PCR
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Total mRNA from the cultured cells was extracted by the Aurum Total RNA Mini Kit (Bio-Rad) according to the manufacturer’s instructions. Total cDNA was synthesized using the Mint revertase (Evrogen, Russia) with miRNA-specific stem-loop primer. After that, real-time PCR was performed with the primers described in the Supplementary Table 1 , and ready-to-use qPCR mix with the SYBR Green I fluorescent dye (Evrogen). Negative controls contained all the components of the PCR mixture but with cDNA replaced by mRNA gave no signal. All PCR reactions were performed using Roche Light cycler 96 real-time detection thermal cycler (Roche, Switzerland). Data was analyzed by the ΔCt method (Livak and Schmittgen, 2001 (link)) using Light-Cycler 96 SW1.01 software (Roche). The expression level of the genes was normalized to the expression level of the housekeeping non-coding RNA U6.
4
Quantitative Real-Time PCR Analysis
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Total mRNA from the tumors and necrotic zones was extracted by the Aurum Total RNA Mini Kit (Bio-Rad) according to the manufacturer’s instructions. cDNA was synthesized using the Mint revertase (Evrogen, Moscow, Russia) with the oligodT primer or miRNA-specific stem-loop primers (Supplementary Table S2 ). After that, real-time PCR was performed with the primers described in the Supplementary Tables S2, S3 , and ready-to-use qPCR mix with the SYBR Green I fluorescent dye (Evrogen). Negative controls contained all the components of the PCR mixture but with cDNA replaced by mRNA gave no signal. All PCR reactions were performed using a Roche Light cycler 96 amplificator (Roche, Basel, Switzerland). PCR reaction was performed in duplicate for every sample and an average was taken for further analysis. Data was analyzed by the ΔCt method (Livak and Schmittgen, 2001 (link)) using Light-Cycler 96 SW1.01 software (Roche). Gene expression level was normalized to expression of the housekeeping genes ACTB, GAPDH, and RPL13A for mRNA or housekeeping non-coding RNA U6 for miRNA analysis.
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