Taqman universal master mix with no amperase uracil n glycosylase

Using the instructions of TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, USA), 10 ng of the total RNA were reverse transcribed. Small nucleus RNA RNU6B, hsa-miR-125b, hsa-miR-425-5p, hsa-miR-21, hsa-miR-200c, hsa-miR-183, hsa-miR-182 and hsa-miR-100 probes and primers were ordered as part of the TaqMan microRNA Assays Kit (Applied Biosystems, USA). cDNA synthesis was performed in a multiplex reaction in which two miRNA primers were used along with the endogenous control (RNU6B). RT-qPCR was performed by means of BioRad CFX384 Real Time System, C1000 Thermal Cycler (Germany). Each well consisted of 5 μL of 2x TaqMan Universal Master Mix with no Amperase Uracil N-glycosylase (UNG) (Applied Biosystems, USA), 2 uL of RNase free water, 0.5 μL of corresponding 20x miRNA probe and 2.5 μL of the cDNA. The reactions were completed in duplicates for each microRNA probe.
Tumor tissues were normalized according to the normal adjacent tissues in the same RT-qPCR run to ensure inter-run calibration. The conditions of cycling were 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and an annealing temperature of 60°C for 60 seconds.
By means of the ΔΔCt equation, the expression of experimental miRNA in tumor tissues was calculated in comparison to the normal adjacent tissue (NAT) samples using the endogenous control RNU6B.

Reverse transcription of 10 ng of RNA was performed using TaqMan^® microRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Multiplex cDNA master mixes were prepared, whereby miR-126 primers were used in each reaction with the endogenous control RNU6B which were part of the TaqMan^® microRNA Assays (Applied Biosystems, USA). RT-qPCR for miR-126 expression was performed using probes that are part of the TaqMan^® microRNA Assays and 2× TaqMan^® Universal Master Mix with no Amperase Uracil N-glycosylase (UNG) (Applied Biosystems, USA) on BioRad CFX96™ or CFX384™ Real-Time PCR Detection System (Hercules, CA, USA). The following steps were run: 10 min hold at 95 °C, 40 cycles of 15 s at 95 °C, and 60 s at 60 °C. miR-126 expression was normalized against the endogenous control RNU6B. Using the ΔΔCq, the relative expression of miR-126 was determined in the tumor samples compared to NAT and in the miR-126 mimic–transfected cells compared to the NC-transfected cells.

For reverse transcription and RT-qPCR, validated primers and probes from TaqMan^® microRNA Assays Kit (Applied Biosystems, Waltham, MA, USA) were used for hsa-miR-16, hsa-miR-21, hsa-miR-19a, hsa-miR-29a, hsa-miR-23a, hsa-miR-145, hsa-miR-203, hsa-miR-155, hsa-miR-210, hsa-miR-31, and hsa-miR-345. cDNA was synthesized for the miRNA of interest starting from 10 ng of extracted RNA using TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. RT-qPCR was then carried out in duplicates for each sample using 2x TaqMan^® Universal Master Mix with no Amperase Uracil N-glycosylase (UNG) (Applied Biosystems, Waltham, MA, USA) as previously described [71 (link)]. RT-qPCR was performed using BioRad CFX96 Real Time System, C1000 Thermal Cycler (Hercules, CA, USA). The cycling conditions were 10 min at 95 °C then 40 cycles of: 15 s at 95 °C and 60 s at 60 °C. The fold change of miRNA expression was calculated using the ∆∆Ct equation where miR-16 is endogenous control and compared to healthy controls.