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qPCR Melt Curve Analysis/ primer specificity
As I am new to qPCR, I am running into a few issues that I don't know how to interpret. Here are my questions:
1.) Can a single primer set be used for multiple purposes? Ex: Can a primer set designed for detecting a gene's expression levels in siRNA transfected cells also be used to detect the gene's expression levels in other various non-transfected cells such as microglia, astrocytes, neurons, whole brain homoginates, as well as be used for genotyping?
Will a primer set designed to detect the effects of siRNA also be used to test the effects of shRNA transfected cells?
We are finding that our primers are working for some purposes, but not others. Ex: A primer works for detecting shRNA but not siRNA in transfected HT22 cells. The same primer set doesn't detect shRNA or siRNA in transfected B65 cells. Is this common? Or are we doing something wrong? Even our transfected controls are not amplifying for these samples. Additionally, I know that the primer set is working because I tested the primer on non transfected microglia cells at the same time on the same plate and the results were exactly as expected.
2.) We are testing out a new primer set. I ran a test plate (control vs mutant) with multiple sample types (astrocytes, microglia, hippocampal slice culture, neurons, and siRNA transfected B65 cells). All the samples amplified, but upon viewing the melt curve results it appears there is a different melt curve Tm for each sample type. Ex: B65 siRNA shows a single melt curve peak at Tm 82 (the primer sets Tm is 82.5), while the control neuron sample shows a single peak at Tm 84. Is this correct? Should each sample type have a different Tm?
I used Beta Actin as my reference gene, and all the samples showed a single distinct melt curve at the correct Tm. Additionally some of the samples really didn't work well at all, such as the microglia, which showed multiple peaks on the melt curve indicating primer dimers. Is there a reason primer dimers might form in one sample type and not another?
Can the varied melt curve peaks for each sample type be explained by primer dimer's of slicing variants? Do you think there is a possible contamination issue?
How far away from the optimal primer Tm is to far for a samples melt curve?
Sooooooo many questions! I hope to receive some helpful answers
|analysis or , curve , melt , melt curve , primer , primer specificity , qpcr , rt-qpcr , specificity|
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