Dear researchers,
I have attempted to do a gene expression study. The first step was isolate the RNA. I have checked the RNA yield using Nanodrop, which had the following nucleic Acid Concentrations (ng/µL)
Gill: 354.7
Kidney: 104.1
Spleen: 39.4
Head kidney: 727.6
Gut wall: 256.4
Water: -0.2
However, when I synthesized cDNA from this RNA the cDNA yield changed to the following:
Gill: 266.8
Spleen: 269.0
Head Kidney: 287.0
Gut wall: 214.5
Kidney: 258.0
Water: 0.5
I was under the impression that the cDNA yield should be the same or very similar to the RNA yields. Therefore, I am finding it tricky to interpret these results.
Any ideas why this could've happened, I would greatly appreciate your input.
Thanks
The two are very different. I dont know the method which you have used for your RT (random hexamer or oligo dT) but I'll give you examples. Your RNA quantified on the Nanodrop contains your mRNA, rRNA, tRNA and small RNA. When you use oligo dT to prepare your RT only the poly adenylated RNA will be reverse transcribed (so only your mRNA), random examer as its name indicates will prime randomly and you can expect all RNA to be reverse transcribed depending if you had a mean to exclude them in your extraction protocol.
Beside you must have (or shoudl have) used the same RNA concentration before doing your RT...so do not expect to see a relation with the amount of RNA.
Yes it does, the oligo dT will 'filter out' rRNA and tRNA. Also you may have tissues which are more metabolically active than other and thus have more mRNA than others.
In our experiments, we only consider RNA concentrations and purity for expression studies. We ignore cDNA amount.
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