MicroRNA quantification was performed by real-time RT-qPCR and miRNA-specific primers and probes (Table 1 ) obtained from Applied Biosystems. Reverse transcriptase (RT) reactions were performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. All RT reactions, no-template controls and RT-minus controls were run in duplicate using TaqMan Universal PCR Master Mix, no AmpErase UNG 2X (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA) and the CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA), as described elsewhere [15 (link)]. The Bio-Rad CFX Manager Software (Bio-Rad, Hercules, CA, USA) was used to collect and analyze data. The relative expression was calculated using the comparative cycle threshold method, and snRNA U6 and hsa-let-7c-5p were used as reference sequences, as previously reported [15 (link),19 (link),20 (link)].
No amperase ung 2x
Manufactured by Thermo Fisher Scientific
Sourced in United States
No AmpErase UNG 2X is a reagent used in real-time PCR applications. It contains a thermostable Uracil-N-Glycosylase (UNG) enzyme that is inactivated during the initial denaturation step of the PCR process. This helps prevent the re-amplification of carryover PCR products from previous experiments.
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Lab products found in correlation
2 protocols using no amperase ung 2x
1
Quantification of miRNA Expression by RT-qPCR
2
miRNA Quantification by RT-qPCR
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MicroRNA relative quantification was performed using real-time RT-qPCR and miRNA-specific primers and probes (reported in Table 1 ) obtained from Applied Biosystems (Thermo Fischer Scientific). Reverse transcriptase (RT) reactions were performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fischer Scientific) according to the manufacturer’s protocol. All RT reactions, including no-template controls and RT-minus controls, were run in duplicate using TaqMan Universal PCR Master Mix, no AmpErase UNG 2X (Applied Biosystems, Thermo Fischer Scientific) and CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA), as described [52] (link). Data were collected and analyzed using Bio-Rad CFX Manager Software Version 1.7 (Bio-Rad). The relative expression was calculated using the comparative cycle threshold method and, as reference, sequences snRNA U6 and hsa-let-7c were used to normalize samples [53] (link).
List of assays employed for miRNA detection.
| miRNA name | Assay ID |
|---|---|
| hsa-miR-93-5p | 001090 |
| hsa U6 snRNA | 001973 |
| hsa-let-7c-5p | 000379 |
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