Difference between revisions of "Team:TecCEM/Results"
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| − | <h1 class = "subTitleUbuntu paddingTop">Diaphorina citri primary cell culture </h1> | + | <h1 class = "subTitleUbuntu paddingTop">Diaphorina citri primary cell culture</h1> |
| − | <p>The main objetive was to develop a primary culture of Diaphorina citri cells, to be transfected with our siRNA and analyzed through flow cytometry. The transfected siRNA sequences were tagged with Alexafluor so they would be visible. This protocol was repeated several times under different conditions, and different compositions of the culture medium were used, which can be seen below. The cells that were cultured in | + | <p>The main objetive was to develop a primary culture of Diaphorina citri cells, to be transfected with our siRNA and analyzed through flow cytometry. The transfected siRNA sequences were tagged with Alexafluor so they would be visible. This protocol was repeated several times under different conditions, and different compositions of the culture medium were used, which can be seen below. The cells that were cultured in Medium A were able to survive after twelve hours, and replicated at a slow rate. Because of this, the flow cytometry was not performed. |
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<p>Figure 1. Cell culture 12 hours after extraction.</br></br></p> | <p>Figure 1. Cell culture 12 hours after extraction.</br></br></p> | ||
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Revision as of 06:29, 1 November 2017
Results
siRNA confirmation
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Sequencing
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Blue BSLA colonies
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Real Time PCR
Since the focus of this project is to verify the effective gene silencing with the four different siRNA we designed, the direct measurement of the amount of mRNA in the cells was required. The first step was to perform a Two-Step Reverse Transcriptase PCR to find out where do the siRNA molecules hybridize with the mRNA. The results are shown below. .
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Table: Expected products for the amplifications in the Two-step RT-PCR.
| Product | Expected size |
|---|---|
| Tubulin | 195 bp |
| Rac I | 193 bp |
| WNT | 200 bp |
| AWD | 180 bp |
| SOD | 199 bp |
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Diaphorina citri primary cell culture
The main objetive was to develop a primary culture of Diaphorina citri cells, to be transfected with our siRNA and analyzed through flow cytometry. The transfected siRNA sequences were tagged with Alexafluor so they would be visible. This protocol was repeated several times under different conditions, and different compositions of the culture medium were used, which can be seen below. The cells that were cultured in Medium A were able to survive after twelve hours, and replicated at a slow rate. Because of this, the flow cytometry was not performed.
Figure 1. Cell culture 12 hours after extraction.
Figure 2. Cell culture 2 days after extraction, observed under an inverted microscope at 10x. No significant cell replication can be observed compared to the first day,
Figure 3. Cell culture 2 weeks after extraction, observed under an inverted microscope at 10x. Cell replication can be observed.
Diaphorina citri laboratory development
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