The Effect of Sweeteners on Nox 4 mRNA and Protein Expression in the Rat Kidney
Location
CSU Ballroom
Start Date
20-4-2015 10:00 AM
End Date
20-4-2015 11:30 AM
Student's Major
Chemistry and Geology
Student's College
Science, Engineering and Technology
Mentor's Name
Theresa Salerno
Mentor's Email Address
theresa.salerno@mnsu.edu
Mentor's Department
Chemistry and Geology
Mentor's College
Science, Engineering and Technology
Description
Dietary sugars have been documented to cause health issues such as obesity, insulin resistance, and hypertension. Because of this, it’s important to study alternative sweeteners. An increase in reactive oxygen species (ROS) can play a prominent role in kidney disease developed from hypertension. The source of the ROS has been linked to the NADPH oxidases (Nox enzymes) that create superoxide radical products. This protein exists as isoforms, with the Nox 4 form being most associated with kidney damage. The purpose of this project was to ask the question whether normotensive rats fed diets supplemented with sugar (sucrose) or sweeteners, stevia and saccharin, exhibit changes in the expression of Nox 4 at both the mRNA and protein levels. Reverse transcription was performed on previously purified RNA, and the Taqman cDNA Reverse Transcription Kit was used to make cDNA templates. Specific primers and a probe were designed to amplify the Nox 4 target and the efficiency of the method was validated. Expression data from the samples were analyzed using the ΔΔCt method to give the expression of Nox 4 relative to the endogenous control, GAPDH. The Western blot was performed by extracting the proteins, measuring total protein content using the BCA Protein Assay Kit, and loading equal amounts of protein onto a 12% Criterion TGX gel. Proteins were transferred to a nitrocellulose membrane and detected using specific primary and IR labeled secondary antibodies. Expression of the Nox 4 protein was measured relative to the amount of GAPDH using a LiCor Odyssey imager.
The Effect of Sweeteners on Nox 4 mRNA and Protein Expression in the Rat Kidney
CSU Ballroom
Dietary sugars have been documented to cause health issues such as obesity, insulin resistance, and hypertension. Because of this, it’s important to study alternative sweeteners. An increase in reactive oxygen species (ROS) can play a prominent role in kidney disease developed from hypertension. The source of the ROS has been linked to the NADPH oxidases (Nox enzymes) that create superoxide radical products. This protein exists as isoforms, with the Nox 4 form being most associated with kidney damage. The purpose of this project was to ask the question whether normotensive rats fed diets supplemented with sugar (sucrose) or sweeteners, stevia and saccharin, exhibit changes in the expression of Nox 4 at both the mRNA and protein levels. Reverse transcription was performed on previously purified RNA, and the Taqman cDNA Reverse Transcription Kit was used to make cDNA templates. Specific primers and a probe were designed to amplify the Nox 4 target and the efficiency of the method was validated. Expression data from the samples were analyzed using the ΔΔCt method to give the expression of Nox 4 relative to the endogenous control, GAPDH. The Western blot was performed by extracting the proteins, measuring total protein content using the BCA Protein Assay Kit, and loading equal amounts of protein onto a 12% Criterion TGX gel. Proteins were transferred to a nitrocellulose membrane and detected using specific primary and IR labeled secondary antibodies. Expression of the Nox 4 protein was measured relative to the amount of GAPDH using a LiCor Odyssey imager.
Recommended Citation
Newman, Logan. "The Effect of Sweeteners on Nox 4 mRNA and Protein Expression in the Rat Kidney." Undergraduate Research Symposium, Mankato, MN, April 20, 2015.
https://cornerstone.lib.mnsu.edu/urs/2015/poster_session_A/38
