These results look great to me. Clear, defined peaks/bands, representing ribosomal RNA.
Oddly, one sample (crab_506) appears to be shifted, relative to the other three, despite exhibiting the same peak/banding pattern. Not sure what would cause something like this; contaminants?
Regardless, we finally have clean RNA and have a usable Bioanalyzer profile to use for reference for crab RNA.
NOTE: The lanes marked with red on the gel representation image indicate that a ribosomal integrity number (RIN) could not be calculated. This is to be expected! The RIN is based on the expectation of two rRNA bands. The anomaly is sample crab_451 – a RIN was actually determined for that sample!
Will likely move forward with additional RNA isolations using the RNeasy Plus Kit (Qiagen).
Firstly, the ladder failed to produce any peaks. Not sure why this happened. Possibly not denatured? Seems unlikely, but next time I run the Pico assay, I’ll denature the ladder aliquot I use prior to running.
Overall, the samples look as they should (see image from TruSeq DNA Methylation Kit manual below), albeit some are a bit lumpy.
Samples from 20091203. 0.5uL was removed from each and transferred to separate tubes and diluted to < 5ng/uL for subsequent Bioanalyzer analysis using the Pico chip. Samples were fragmented using RNase III according to the Ambion WTK protocol and then cleaned up/concentrated using the Invitrogen RiboMinus Concentration Module according to the Ambion WTK protocol.
Samples were spec’d prior to running on the Bioanalyzer:
Concentrations/absorbance values are not accurate when using the NanaDrop after using the RiboMinus Concentration module, according to the Ambion WTK protocol. However, yields seem pretty good…
Total, mRNA and fragmented mRNA from each of the four samples was run on the Pico chip with the Eukaryote Total RNA Bioanalyzer protocol.
The 2L tot (total RNA) and 3L tot (total RNA) samples are clearly very good quality. 2L tot does exhibit some very slight degradation, though. 4L tot (total RNA) and 6L tot (total RNA) show a much greater degree of degradation. All mRNA samples show complete removal of any trace, contaminating rRNA. The fragmented samples (the last four samples on the gel image above) all appear to be perfect. The 4L frag sample simply has less RNA loaded and that is why it is not as dark as the other three fragmented samples. Despite the degradation in the 4L tot and 6L tot samples, the fragmentation profile looks good and we will proceed with making the cDNA libraries for those samples.