Hello,
I am a Master's student working with Murine Skin samples for qPCR analysis. I am having a hard time getting clean ribosomal bands with my RNA- some appear but are still VERY smeared. I have good A260/280 ratios (around 2). I am homogenizing using a drill and lysis buffer + Proteinase K for 30-45 minutes. I then pull off the supernatant and isolate my RNA.
I have attached an example of what my bands look like. I seem to be getting good amplification from the cDNA made from the RNA, but I would like to clean it up before I publish with it.
Thank you in advance!
Thank you for your response- that paper has been really helpful.
I am not sure where in my process that the RNA is degrading. I am homogenizing using a drill bit then extracting using Zymo's Miniprep plus kit.
My samples were stored in RNALater so I have been rinsing them with nuclease free water before homogenizing.
Degradation of nucleic acids is usually caused by non-optimal sample conditions. Due to endogenous nucleases present in the sample (such as RNase), the integrity of nucleic acid extracts is bound to decrease through time. To achieve better results, it would be best to immediately process tissues for extraction and add RNase inhibitors during sample preparation.
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