Methods for Extracting RNA and Protein from a Single Tissue Sample
Mentor 1
James Moyer
Location
Union Wisconsin Room
Start Date
29-4-2016 1:30 PM
End Date
29-4-2016 3:30 PM
Description
When introducing a novel chemical compound into a biological system, the response of that system is determined through analyses of downstream signaling molecules associated with biochemical changes in the system. Our lab has demonstrated that intrahippocampal administration of the calcium-binding protein apoaequorin (AQ) protects neurons from an ischemic insult. This effect is time-dependent and may involve a neuroimmunomodulatory component, as evidenced by measurable changes in cytokine and chemokine RNA associated with the AQ administration (Detert et al., 2013). To date, our lab has analyzed extracted tissues through either a protein extraction (followed by Western blot analysis) or an RNA extraction (followed by rtPCR). Thus, our samples were limited to one of the methods of analysis, which meant that different animals had to be used for each analysis. In an effort to directly relate changes in both protein and RNA within the same animal, we investigated two different protocols that use two different reagents. Our goal was to develop a procedure that allowed us to extract quality RNA and protein from the same tissue sample. Following extraction of RNA and proteins, qualitative and quantitative results were obtained that assessed our ability to analyze cytokine and chemokine gene and protein expression. Our data suggest that we can use one reagent and one method to simultaneously extract both RNA and protein with sufficient yield of both. This will greatly enhance our ability to directly relate changes in both RNA and protein within a single animal as a function of any of a wide range of experimental manipulations or treatment conditions.
Methods for Extracting RNA and Protein from a Single Tissue Sample
Union Wisconsin Room
When introducing a novel chemical compound into a biological system, the response of that system is determined through analyses of downstream signaling molecules associated with biochemical changes in the system. Our lab has demonstrated that intrahippocampal administration of the calcium-binding protein apoaequorin (AQ) protects neurons from an ischemic insult. This effect is time-dependent and may involve a neuroimmunomodulatory component, as evidenced by measurable changes in cytokine and chemokine RNA associated with the AQ administration (Detert et al., 2013). To date, our lab has analyzed extracted tissues through either a protein extraction (followed by Western blot analysis) or an RNA extraction (followed by rtPCR). Thus, our samples were limited to one of the methods of analysis, which meant that different animals had to be used for each analysis. In an effort to directly relate changes in both protein and RNA within the same animal, we investigated two different protocols that use two different reagents. Our goal was to develop a procedure that allowed us to extract quality RNA and protein from the same tissue sample. Following extraction of RNA and proteins, qualitative and quantitative results were obtained that assessed our ability to analyze cytokine and chemokine gene and protein expression. Our data suggest that we can use one reagent and one method to simultaneously extract both RNA and protein with sufficient yield of both. This will greatly enhance our ability to directly relate changes in both RNA and protein within a single animal as a function of any of a wide range of experimental manipulations or treatment conditions.
