So what would 25 Haverford College Biology majors be doing in a dimly lit laboratory right before Thanksgiving break? Synthesizing cDNA and creating labeled targets, of course!
The goal for this week was to transcribe cDNA from our RNA samples. But when we transcribed our labeled targets, we wanted to be able to distinguish one sample from the other. Therefore, we used two different cyanine fluorescent dyes as part of our cDNA synthesis to help us visualize our unique samples on the microarray.
But why were we working in the dark? (There was a reason for the madness). The cyanine dyes that we are using are sensitive to light and begin to degrade when exposed to natural or fluorescent light. And so, we wanted to use minimal lighting to synthesize our cDNA using cyanine labeled dNTPs.
dNTPs are dinucleotide triphosphates which provide the bases when transcribing cDNA from RNA. Each dye was specific to a sample so that we would be able to distinguish one sample from the other.
So for our circadian rhythm group, the Cy3 labeled samples will light up red for genes that are expressed in the light period and the Cy5 labeled samples will light up green for genes that are expressed in the dark period. For genes that are expressed in both conditions, we will see a yellow dot.