when I extracted four different samples, I was able to obtain 24 ng/uL for one of the samples but nothing at all for the other samples. All these were obtained from the same person and the exact same protocol was followed.
How are you quantifying your RNA? Is your method for quantification reliable?
As long as you'requested following manufacturer protocols, the kit is reliable - I use the same one. You have one or both of two problems: RNase contamination or unreliable detection.
Thank you Antonio and Carl for your inputs.
I used RNAzap to clean all the pipettes, lab bench, outside of pipette tip box, racks and outside of the tubes that hold the reagents. Also, I used RNAse free tubes and pipette tips. So, the chances of RNAse contamination is very minimal.
I used Qubit RNA HS assay and Nanodrop to quantify my RNA. With Qubit, it showed the concentrations were too low for three of the samples. However, with Nanodrop, the concentrations of three samples were around 6- 7 ng/uL and the fourth sample was 26 ng/uL. All four samples had a 260/280 ratio between 2.01 and 2.35. This is where I am confused. Because, Qubit can measure low levels of RNA and I use nanodrop to check the purity. But, the concentration difference for the fourth sample between qubit and nanodrop is ~2ng/uL. So, I am not sure if I could take in the concentrations given by nanodrop for the other three samples or if I should redo the extraction considering Qubit could not recognize the RNA in those samples.
Sincerily, I do not trust Nanovue. We do not use it anymore in our lab except for purity.
All samples were collected and stored in the same way? Maybe that is your problem, sample preservation.
I second that. We just use Nanodrop to check the purity and use Qubit to assess the concentration.
All the samples were preserved in RNALater and stored at -20C and I worked on all the samples at the same time, within hours of collection. It probably boils down to the way I yanked out the hair samples. I probably disrupted the follicles when I pulled the hairs for three of my samples. So, it probably yielded insignificant amount of RNA which probably might have been overpowered by salts in the elute.
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