Homologs are more important in terms of the primers and sequence we’re trying to amplify. You want the least amount of homologs, ideally zero, to ensure your primers bind uniquely to the sequence of interest and not to any other parts of the tetrahymena genome.
Not possible for our primers necessarily as we design them to be about 20 bases and not have any extraneous components or characteristics. However if they amplified an area that had introns in the genomic DNA, this would cause a size difference between our cDNA(no introns) and gDNA bands in the gel. It’s very fortunate our primers annealed to an area containing no introns so we knew our area of amplification would be the same for both and any differences would be the result of a mistake or contamination.
We used an image analysis program called Fiji that was able to compare light intensity within the image to help us compare the two bands. therefore giving us a quantitative number(130%) for how much more intense the treated band was compared to the untreated.
You said that there was 130% expression in DCR-2 from induced damage. Do you have a rough guess on what the expression value would be in nature? I imagine it would vary depending on how the DNA was damaged?
You are correct that it would vary, In nature there are a multitude of forms in which DNA damage occurs so we can’t say for sure what that would look like. Under induced conditions DNA damage is done to an extreme that we would not see naturally so we’re rea;;y comparing as natural as we can achieve in the lab vs induced conditions to see if there is upregulation of the gene. If I had to give an estimate I’d say there is probably still upregulated expression under more severe DNA damaging conditions in natural environments, but it is near impossible to study this and give conclusive results. All we can say for sure from this research is that DCR-2 is upregulated in expression in the presence of DNA damage which tells us it contributes in some way to the repair pathway. Great question!
It was purely a primer validation, we wanted to see if the primers we selected would actually work to amplify the gene of interest and be useful in future experiments. We had to make sure these primers were valid so we could go forward with amplifying our gene under untreated and treated conditions and measure expression using them.
curesymposium.org 
What are homologs, and how does their presence affect the behavior of the target bacteria?
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Homologs are more important in terms of the primers and sequence we’re trying to amplify. You want the least amount of homologs, ideally zero, to ensure your primers bind uniquely to the sequence of interest and not to any other parts of the tetrahymena genome.
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What would happen if the primers had introns? Is that chemically possible?
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Not possible for our primers necessarily as we design them to be about 20 bases and not have any extraneous components or characteristics. However if they amplified an area that had introns in the genomic DNA, this would cause a size difference between our cDNA(no introns) and gDNA bands in the gel. It’s very fortunate our primers annealed to an area containing no introns so we knew our area of amplification would be the same for both and any differences would be the result of a mistake or contamination.
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How did you analyze/measure the intensity of the bands in Figure 2A to collect your quantitative data?
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We used an image analysis program called Fiji that was able to compare light intensity within the image to help us compare the two bands. therefore giving us a quantitative number(130%) for how much more intense the treated band was compared to the untreated.
LikeLike
You said that there was 130% expression in DCR-2 from induced damage. Do you have a rough guess on what the expression value would be in nature? I imagine it would vary depending on how the DNA was damaged?
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You are correct that it would vary, In nature there are a multitude of forms in which DNA damage occurs so we can’t say for sure what that would look like. Under induced conditions DNA damage is done to an extreme that we would not see naturally so we’re rea;;y comparing as natural as we can achieve in the lab vs induced conditions to see if there is upregulation of the gene. If I had to give an estimate I’d say there is probably still upregulated expression under more severe DNA damaging conditions in natural environments, but it is near impossible to study this and give conclusive results. All we can say for sure from this research is that DCR-2 is upregulated in expression in the presence of DNA damage which tells us it contributes in some way to the repair pathway. Great question!
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Why were primers used in 1A and how did it contribute to the research?
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Why did you use primers in 1A and how does it contribute to the research?
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It was purely a primer validation, we wanted to see if the primers we selected would actually work to amplify the gene of interest and be useful in future experiments. We had to make sure these primers were valid so we could go forward with amplifying our gene under untreated and treated conditions and measure expression using them.
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