RiboMinus™ Transcriptome Isolation Kit, bacteria

This is most likely due to contamination of the total RNA sample with genomic DNA. Before the RiboMinus Kit procedure, we recommend treating the total RNA sample with DNase I to remove any genomic DNA contamination.

The purified RNA is easily quantitated using UV absorbance at 260 nm or a Quant-iT RNA Assay Kit. To verify rRNA depletion, electrophorese your sample on an agarose gel or use an Agilent 2100 Bioanalyzer. Agarose gel electrophoresis should show depletion of 18S and 28S rRNA bands compared to a control sample. Absence of contaminating DNA and RNA degradation may also be confirmed by agarose gel electrophoresis. The efficiency of rRNA depletion in the purified RNA, RNA degradation, and RNA concentration can also be analyzed using a bioanalyzer.

With regard to species specificity, the following organisms have 0 mismatches for the LSU/SSU probes (LSU (28S) / SSU (18S) Probe perfect matches). This means the probes will remove the 18S and 28S sequences from the following species.

- Species from the Land: Human, mouse, rat, frog, rabbit, cow, pig, chicken, Drosophila,* Caenorhabditis elegans, midge, mosquito, yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe)
- Species from the Sea: Sea squirt, coelacanth, lancelet, eel, salmon, sturgeon, ratfish, lamprey, brown plankton, zooplankton
- Plants: Arabidopsis thaliana, Brassica napus, rice, tomato, Humulus lupulus, Zea mays, wheat, soybean, pine, aspen

* The 28S and 18S probes in this kit respectively align 100% to the Drosophila 18S and 28S rRNA sequence. However, there are reports suggesting Drosophila (insect) 28S rRNA split into 2 smaller fragments co-migrating with 18S. The exact split site is not identified yet. But based on customer feedback, this kit does not remove the split 28S rRNA completely. We have not tested Drosophila samples with this kit.

The 5S and 5.8S sequences are more divergent, and thus the best probes possible have reduced species breadth:
- 5S: zero to 1 mismatch: human, mouse, rat, frog, chicken, Drosophila, C. elegans, A. thaliana, Z. mays
- 5.8S: zero mismatches: human, mouse, rat