Briefly, cells were pre-treated with SDG (50 μM, 6 h) and irradiated (2 Gy). Total RNA was isolated from using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). qPCR was performed using 25 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA and calibrated to untreated control samples according to the ΔΔCT method as shown previously [22 (link)].
Quantitative analysis of antioxidant gene expression
Briefly, cells were pre-treated with SDG (50 μM, 6 h) and irradiated (2 Gy). Total RNA was isolated from using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). qPCR was performed using 25 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA and calibrated to untreated control samples according to the ΔΔCT method as shown previously [22 (link)].
Corresponding Organization : University of Pennsylvania
Protocol cited in 3 other protocols
Variable analysis
- SDG treatment (50 μM, 6 h)
- MRNA expression of antioxidant genes (heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase, quinone 1 (NQO1), and glutathione S-transferase mu 1 (GSTM1))
- Irradiation (2 Gy)
- RNA isolation and quantification
- Reverse transcription of RNA to cDNA
- QPCR assay conditions (25 ng of cDNA per reaction well, StepOnePlus™ Real-Time PCR System)
- Gene expression data normalization (to 18S ribosomal RNA) and calibration (to untreated control samples)
- Untreated control samples
Annotations
Based on most similar protocols
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!