RNA was isolated using Trizol and the RNeasy micro kit with on-column deoxyribonuclease I treatment (Qiagen). Quality and integrity of RNA was determined using nanodrop spectrophotometer ND-1000. Reverse-transcriptase reactions were prepared using 1 µg of RNA and iScript cDNA Synthesis Kit (Bio-Rad). Quantitative real-time PCR was completed using TaqMan probes (Applied Biosystems) for Gcg (Mm01269055_m1), Agrp (Mm00475829_g1), Kiss1 (Mm03058560_m1), and housekeeping gene 18s (Hs03003631_g1) was used as an endogenous control to normalize each sample and gene. PCRs were in a 10-μl volume using 0.5 μl TaqMan probe, 10 ng cDNA template, 5 μl TaqMan Gene Expression Master Mix II with UNG (Applied Biosystems), and 2.5 μl DNase/RNase-free molecular grade water (Qiagen). Real-time PCR was run using an Applied Biosystems 7900HT Fast Real-Time PCR system with initial denaturing at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and annealing at 60°C for 1 min. Results were calculated using the Pfaffl method (Pfaffl, 2001 (link)).
Taqman gene expression master mix 2 with ung
Manufactured by Thermo Fisher Scientific
TaqMan Gene Expression Master Mix II with UNG is a ready-to-use solution for real-time PCR gene expression analysis. It contains a hot-start DNA polymerase, dNTPs, and a uracil-N-glycosylase (UNG) enzyme for carryover contamination prevention.
Automatically generated - may contain errors
Lab products found in correlation
Taqman probe, by Thermo Fisher Scientific (4 mentions)
Deoxyribonuclease 1 treatment, by Qiagen (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Cdna synthesis kit, by Promega (1 mentions)
2 protocols using taqman gene expression master mix 2 with ung
1
Quantifying Gene Expression with Real-Time PCR
2
Quantifying Gene Expression in Murine Hypothalamus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hypothalami were collected from control and DIO mice. Tissue was homogenized in Trizol and RNA was isolated using the RNeasy micro kit with on-column deoxyribonuclease I treatment (Qiagen). Quality and integrity of RNA was determined using a ND-1000 Nanodrop spectrophotometer. Reverse-transcription reactions were prepared using 2 μg of RNA and a cDNA Synthesis Kit (Promega). Quantitative real-time PCR was performed using TaqMan probes (Applied Biosystems) for RELN (Mm00465200_m1), ApoER2 (Mm00474030_m1), and VLDLR (Mm00443298_m1). The level of 18s rRNA (Hs03003631_g1) was used as an endogenous control for normalization. PCRs were performed in a 10-μl volume using 0.5 μl TaqMan probe, 20 ng cDNA template, 5 μl TaqMan Gene Expression Master Mix II with UNG (Applied Biosystems), and 2.5 μl DNase/RNase molecular-grade water (Qiagen). Real-time PCR was run using a 7900HT Fast Real-Time PCR system with initial denaturation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and annealing at 60 °C for 1 min.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!