Total RNA was extracted from cell cultures with TRI-zol reagent. Five micrograms of total RNA was reverse transcribed using the Moloney Murine Leukemia Virus Reverse transcriptase (M-MuLV) (New England BioLabs Inc., Ipswich, MA, USA). RT-PCR was performed with 1 μL of cDNA using the TaqMan™ detection system (Thermo Fisher Scientific) and the Universal PCR Master Mix reagents kit (Thermo Fisher Scientific). Probes previously developed from TaqMan were used to study Eag1 (ID: Hs00924320_m1) and Gusb (ID: Hs00939627_m1, as a constitutive gene) expression. The PCR reaction protocol was 95°C for 15 seconds and 60°C for 1 minute (40 cycles). Data were analyzed with the 2−ΔΔCt method.
Universal pcr master mix reagents kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Universal PCR Master Mix is a reagent kit designed for use in polymerase chain reaction (PCR) amplification. The kit contains a pre-formulated mix of necessary components, including DNA polymerase, buffers, and nucleotides, to perform PCR reactions.
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Lab products found in correlation
2 protocols using universal pcr master mix reagents kit
1
Eag1 Expression Analysis by RT-PCR
2
Quantifying KCNH1 Gene Expression
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Total RNA was extracted with TRIzol reagent. Two micrograms of total RNA was reverse-transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MuLV, M0253S, BioLab®, Inc., Lawrenceville, GA, USA). Real-time PCR was performed with 1 µL of cDNA using the TaqMan™ detection system (Applied Biosystem, Foster City, CA, USA) and the Universal PCR Master Mix reagents kit (Thermo Fisher Scientific, Waltham, MA, USA). KCNH1 TaqMan probes for Eag1 (ID: Hs00924320_m1) and h-HPRT (hypoxanthine-guanine phosphoribosyl transferase, Part Number 4326321E, constitutive gene) were used. The PCR protocol was 95 °C for 15 s and 60 °C for 1 min (40 cycles). The data were analyzed using the 2−∆∆Ct method.
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