500 ng of total RNA was subjected to reverse transcription according to the protocol of the RevertAid First-Strand Synthesis Kit (K1622, TFS). Transcript quantification was performed by qPCR with Maxima SYBR Green/ROX qPCR Master Mix (K0223, TFS) on the CFX Connect Thermal Cycler System (Bio-Rad). Target gene levels were normalized to the housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were transformed as described previously [21 (link)]. Forward and reverse primers used in experiments are depicted in Table S1. If not indicated differently, results are presented as mean ± SEM for two independent biological repeats. Graphs are prepared using GraphPad Prism 8.3.0.
Total rna zol out d kit
The Total RNA Zol-Out™ D kit is a reagent designed for the isolation and purification of total RNA from biological samples. It utilizes a guanidinium-based extraction method to efficiently separate RNA from other cellular components.
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6 protocols using total rna zol out d kit
Quantifying Transcript Levels by RT-qPCR
500 ng of total RNA was subjected to reverse transcription according to the protocol of the RevertAid First-Strand Synthesis Kit (K1622, TFS). Transcript quantification was performed by qPCR with Maxima SYBR Green/ROX qPCR Master Mix (K0223, TFS) on the CFX Connect Thermal Cycler System (Bio-Rad). Target gene levels were normalized to the housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were transformed as described previously [21 (link)]. Forward and reverse primers used in experiments are depicted in Table S1. If not indicated differently, results are presented as mean ± SEM for two independent biological repeats. Graphs are prepared using GraphPad Prism 8.3.0.
RNA Isolation and Expression Analysis
Quantification of FMR1 mRNA and pre-mRNA Levels
Total RNA Isolation and Reverse Transcription
Total RNA Extraction from Mouse Tissues
RNA Extraction and qPCR Analysis
Inhibitor, RevertAid and dNTP Mix according to the manufacturer's protocol (Thermo Scientific, USA). Real-time qPCR (RT-qPCR) was performed in triplicates with 5xHOT FIREPol EvaGreen qPCR Supermix (Solis BioDyne, Estonia) and the primers from TAG Copenhagen (Supplementary Table S1) using ViiA™ 7 Real-Time PCR system (Applied Biosystems, USA). The relative gene expression levels were normalized according to the level of elongation factor 1 alpha (EF1A) in case of cell cultuers and beta-2 microglobulin (B2M) in case of mouse samples J o u r n a l P r e -p r o o f and calculated using the comparative Ct (ΔΔCt) method. The mean level of non-transfected cells or control experiments was equalized to 1.
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