Quantitative RT-PCR (qRT-PCR) was performed on a Step-One-Plus real-time PCR machine (Applied Biosystems) and analyzed by the comparative CT method. Amplification was monitored using PerfeCTa SYBR Green SuperMix with ROX (Quanta Bio). Analysis of TMV miRNA levels was performed using miR specific primers purchased from Quanta Bio: hsa-miR-21–5p HSMIR-0021–5P; hsa-miR-100–5p HSMIR-0100–5P; hsa-miR-27a-3p HSMIR-0027A-3P; and hsa-miR-151a-3p HSMIR-0151A-3P. Pre-miRNA levels were analyzed using the same workflow, with the primers listed above. qRT-PCR to determine miR-21 knockdown was carried out as described above using Taqman probe (Hs04231424_s1) from ThermoFisher. EGFP (Mr04097229_mr) and SMAD-7 (Hs00998193_m1) levels were measured using the Fast Advanced Cells to CT kit from ThermoFisher. All Taqman assays used 18S ribosomal RNA (Hs99999901_s1) as input control and were cycled according to manufacturer’s instructions.
Fast advanced cells to ct kit
Manufactured by Thermo Fisher Scientific
The Fast Advanced Cells to CT kit is a laboratory tool designed for the rapid and efficient extraction of cellular RNA from samples. It provides a straightforward process for preparing RNA samples for downstream analysis, such as real-time PCR or gene expression studies.
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2 protocols using fast advanced cells to ct kit
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Quantitative RT-PCR Analysis of miRNA Levels
2
Quantitative RT-PCR Analysis of miRNA Levels
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Quantitative RT-PCR (qRT-PCR) was performed on a Step-One-Plus real-time PCR machine (Applied Biosystems) and analyzed by the comparative CT method. Amplification was monitored using PerfeCTa SYBR Green SuperMix with ROX (Quanta Bio). Analysis of TMV miRNA levels was performed using miR specific primers purchased from Quanta Bio: hsa-miR-21–5p HSMIR-0021–5P; hsa-miR-100–5p HSMIR-0100–5P; hsa-miR-27a-3p HSMIR-0027A-3P; and hsa-miR-151a-3p HSMIR-0151A-3P. Pre-miRNA levels were analyzed using the same workflow, with the primers listed above. qRT-PCR to determine miR-21 knockdown was carried out as described above using Taqman probe (Hs04231424_s1) from ThermoFisher. EGFP (Mr04097229_mr) and SMAD-7 (Hs00998193_m1) levels were measured using the Fast Advanced Cells to CT kit from ThermoFisher. All Taqman assays used 18S ribosomal RNA (Hs99999901_s1) as input control and were cycled according to manufacturer’s instructions.
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