Gene expression of p53, p21 and MDM2 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). After treatment at IC50 concentrations as described above, total RNA was extracted with the Ambion Minikit (Ambion, Carlsbad, CA) and cDNA was synthesized (High Capacity cDNA Archive kit, Applied Biosystems, Carlsbad, CA) according to manufacturer’s instructions. Using TaqMan primer sets for p53, p21 (cyclin-dependent kinase inhibitor 1), and MDM2, qRT-PCR was performed in triplicate with the housekeeping gene cyclophilin (PPIA; Applied Biosystems) as normalizer. The Bio-Rad C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) was used for all reactions, and fold-change was calculated with the 2-△△Ct method.
Cyclophilin
Manufactured by Thermo Fisher Scientific
Cyclophilin is a type of protein found in various organisms, including humans. It plays a role in the folding and stabilization of other proteins. Cyclophilin is a commonly used tool in biochemical research and assays.
Automatically generated - may contain errors
Lab products found in correlation
C1000 thermal cycler, by Bio-Rad (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Microamp optical 96 well reaction plate, by Thermo Fisher Scientific (1 mentions)
Sds v2, by Thermo Fisher Scientific (1 mentions)
Taqman pcr core reagent kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Taqman qpcr, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
3 protocols using cyclophilin
1
Quantitative Analysis of p53, p21 and MDM2 Expression
2
Quantifying ADAM Metalloproteinase Expression
3
Quantifying RAGE mRNA Expression in Kidney
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Total RNA was extracted from the kidney using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The SuperScript II First-Strand Synthesis System for real-time PCR (Invitrogen) kit was utilized for the synthesis of 20 μl of complementary DNA from 1000 ng of total RNA. The mRNA levels of RAGE (Applied Biosystems, Thermo Fisher Scientific Inc., Foster City, CA, USA) were determined by real-time PCR. Quantitative measurements were made using a commercial kit (TaqMan qPCR; Applied Biosystems) in a detection system (StepOne Plus; Applied Biosystems). Cycling conditions were as follows: enzyme activation at 50 °C for 2 min, denaturation at 95 °C for 10 min, complementary DNA products amplification for 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Gene expression was quantified in relation to the values of the control group after normalization to an internal control (cyclophilin; Applied Biosystems).
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