Hi All,
I am at my wits end and was hoping someone might provide some insight. I am working on a transcriptomic project with microfilariae (immature filarial nematodes) and have been using Trizol and the Zymo Research Clean & Concentrate kit. I have been struggling on a few fronts:
1) When I first look at my RNA on the Nanodrop, everything looks nearly perfect, 260/280 consistently higher than 1.85, 260/230 pretty good as well, with high concentrations (500+ ng/ul), peaks obviously look good
2) When I send my samples off for the Bioanalyzer, I am consistently getting large differences in concentration both higher and lower, but mostly lower
Example: Nanodrop=457 Bioanalyzer= 66 or Nanodrop= 619.6 Bioanalyzer=77
3) Bad RIN scores: I am fully aware that I likely have degraded samples, but I was wondering if the Bioanalyzer might have an issue with the sample type itself. These parasites went through a mosquito and into a gerbil before I collected them, so I am wondering if it might be like the issues we see with insect or fungi samples? I have been getting lots of 2-3 scores and only a handful higher than 5 (after 50+ sample submissions with 4 different treatment groups).
I would appreciate any feedback you guys might have, and if this is not the correct forum or you suggest cross-posting, just let me know
Thanks,
Mary
I am at my wits end and was hoping someone might provide some insight. I am working on a transcriptomic project with microfilariae (immature filarial nematodes) and have been using Trizol and the Zymo Research Clean & Concentrate kit. I have been struggling on a few fronts:
1) When I first look at my RNA on the Nanodrop, everything looks nearly perfect, 260/280 consistently higher than 1.85, 260/230 pretty good as well, with high concentrations (500+ ng/ul), peaks obviously look good
2) When I send my samples off for the Bioanalyzer, I am consistently getting large differences in concentration both higher and lower, but mostly lower
Example: Nanodrop=457 Bioanalyzer= 66 or Nanodrop= 619.6 Bioanalyzer=77
3) Bad RIN scores: I am fully aware that I likely have degraded samples, but I was wondering if the Bioanalyzer might have an issue with the sample type itself. These parasites went through a mosquito and into a gerbil before I collected them, so I am wondering if it might be like the issues we see with insect or fungi samples? I have been getting lots of 2-3 scores and only a handful higher than 5 (after 50+ sample submissions with 4 different treatment groups).
I would appreciate any feedback you guys might have, and if this is not the correct forum or you suggest cross-posting, just let me know
Thanks,
Mary
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