TY - GEN
T1 - Optimization of siRNA transfection into Human Umbilical Vein Endothelial Cells and Aortic Smooth Muscle Cells for the knockdown of zinc finger protein 496 expression
AU - Amanuel, Teklu,
N1 - Optimization of siRNA transfection into Human Umbilical Vein Endothelial Cells and Aortic Smooth Muscle Cells for the knockdown of zinc finger protein 496 expression
Teklu, Amanuel
Örebro University, School of Health and Medical Sciences.
2011 (English)
Independent thesis Advanced level (degree of Master (One Year)), 10 credits / 15 HE credits
Student thesis
Place, publisher, year, edition, pages
2011.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:oru:diva-17351
ISRN: ORU-HAK/MED-AS-2011/0011--SE
OAI: oai:DiVA.org:oru-17351
DiVA: diva2:443545
Subject / course
Medicine
Uppsok
Medicine
Supervisors
Sirsjö, Allan
Available from: 2011-09-27 Created: 2011-09-26 Last updated: 2012-09-03
Bibliographically approved
Open Access in DiVA
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Professor Allan Sirsjo ([email protected]) was a supervisor for the master's degree project.
PY - 2011/9/27
Y1 - 2011/9/27
N2 - Background: Zincfinger protein 496 is a DNA-binding transcription factor and its function is largely unknown.Gene knockdown using small interfering RNAs is highly efficient technology to study gene functions.Maximum knock-down of target gene merely depends on the design and efficient delivery of small interfering RNA into cells in optimised transfection conditions.The aim of this study was optimization of small interfering RNA transfection into aortic smooth muscle cells and human umbilical vein endothelial cells for silencing zinc finger protein 496expression.Method:Aortic smooth muscle cells and human umbilical vein endothelial cells from passage 5 to10 were transfected with different concentrations of target-specificZNF496siRNA and non-target specific siRNA negative control using lipofectamine2000 carrier molecule. Expressions of ZNF496 in the transfected cells were done using quantitative real-time PCR. Result: Reductions of ZNF496 expression were obtained in both HUVEC and AoSMC cells. All the different concentrations of ZNF496 siRNA (10, 20, 40, 80, 160 pmol) resulted in a reduction of ZNF496 expression in HUVEC cells, but only 10pmol and 80pmol ZNF496 siRNA concentrations induced a reduction in AoSMC. There was up to four-fold reduction of ZNF496 expression in HUVEC cells transfected with target specific ZNF496 siRNAcompared to HUVEC cells transfected with siRNA negative control, whereas in AoSMC, there was only up to two-fold reduction in cells transfected with ZNF496 siRNA compared to cells transfected with siRNAnegative control.Conclusion:Optimization of siRNA concentration which results in reduction of ZNF496 expression was performed. This result will be used for functional studies of ZNF496 and its associated genes.
AB - Background: Zincfinger protein 496 is a DNA-binding transcription factor and its function is largely unknown.Gene knockdown using small interfering RNAs is highly efficient technology to study gene functions.Maximum knock-down of target gene merely depends on the design and efficient delivery of small interfering RNA into cells in optimised transfection conditions.The aim of this study was optimization of small interfering RNA transfection into aortic smooth muscle cells and human umbilical vein endothelial cells for silencing zinc finger protein 496expression.Method:Aortic smooth muscle cells and human umbilical vein endothelial cells from passage 5 to10 were transfected with different concentrations of target-specificZNF496siRNA and non-target specific siRNA negative control using lipofectamine2000 carrier molecule. Expressions of ZNF496 in the transfected cells were done using quantitative real-time PCR. Result: Reductions of ZNF496 expression were obtained in both HUVEC and AoSMC cells. All the different concentrations of ZNF496 siRNA (10, 20, 40, 80, 160 pmol) resulted in a reduction of ZNF496 expression in HUVEC cells, but only 10pmol and 80pmol ZNF496 siRNA concentrations induced a reduction in AoSMC. There was up to four-fold reduction of ZNF496 expression in HUVEC cells transfected with target specific ZNF496 siRNAcompared to HUVEC cells transfected with siRNA negative control, whereas in AoSMC, there was only up to two-fold reduction in cells transfected with ZNF496 siRNA compared to cells transfected with siRNAnegative control.Conclusion:Optimization of siRNA concentration which results in reduction of ZNF496 expression was performed. This result will be used for functional studies of ZNF496 and its associated genes.
M3 - Other contribution
PB - DiVA
ER -