Hello!
I'm a M.S. student at the University of North Carolina at Charlotte studying fiddler crabs and their embryos/larvae. We've been preserving our samples using RNAlater throughout the summer and I'm now extracting them. I've determined that the RNAqueous Micro Kit is the best one for us to use, and I'm using a NanoDrop for quantification.
I did three extractions from the same clutch, each with 50 larvae. My nanodrop concentrations were as follows: 146.7 ng/uL, 154.4 ng/uL, and 126.5 ng/uL. My 260/280 for each was 1.99, 1.96, 1.98, which from my understanding is good. However, my 260/230 were -1.04, -1.74, -1.12.
I have two questions:
1. Are these RNA concentrations high enough to be sent off for RNA sequencing? I'm new to the world of RNA sequencing so any advice is appreciated.
2. What would cause my 260/230 values to be negative? My understanding is that they should be close to 2.
Thank you!
Hey Caitlin! I recently did a tutorial zoom call for RNA-Seq with Tecan and they mentioned your ND concentrations should be 300 ng/uL or better. That could be for their specific kit, but I would imagine higher numbers are better, given that you will lose quite a bit of sample during all of the wash steps. Also, your 260/230 values can show negative if the instrument wasn't cleaned properly by the previous user/between samples. Alternatively, you could have a bubble in your sample. In the past, when I have had weird reads, I loaded 1.5 uL instead of 1 uL and really watched for bubbles, but that's totally up to you. Good luck!
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