1. How does the Ribo-Zero procedure work?
Ribo-Zero kits remove ribosomal RNA (rRNA) using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The probe:rRNA hybrid is then captured by magnetic beads and removed using a magnet, leaving the desired rRNA-depleted RNA in solution.
2. Do you still sell the older, nonmagnetic Ribo-Zero kits?
Nonmagnetic Ribo-Zero Kits were discontinued effective June 30, 2012.
3. How much total RNA can be used with the Ribo-Zero Kits?
The normal range of input total RNA is 1-5 µg. Attempts to add more than the recommended mass risks saturation of the rRNA removal probes, thus increasing the amount of rRNA in the depleted RNA sample.
4. Will a Ribo-Zero kit work with (insert species here) RNA?
Ribo-Zero kits will remove rRNA from a broad variety of bacteria, fungi/yeast, plants, and eukaryotic total RNA samples. We can provide empirical information on the homology of the rRNA of interest to the Ribo-Zero capture probes, and thus provide an estimate of the ability of a specific Ribo-Zero kit to remove rRNA from nearly any sample. Please contact Epicentre Tech Support for further information.
5. How well do Ribo-Zero kits work with yeast?
The Ribo-Zero Gold and Human/Mouse/Rat Kits are able to remove the majority of yeast or fungal rRNA, depending on the relative homologies between the yeast rRNA and the capture probes. Species of yeast and fungi tested successfully at Epicentre include Saccharomyces cerevisiae, S. pombe, Candida, Aspergillus, and others.
6. What is the difference between the standard Ribo-Zero Kit and the Ribo-Zero Gold Kit (Human/Mouse/Rat)?
The Ribo-Zero Gold Kit contains additional capture probes that cover mitochondrial frRNA sequences, which account for about 7% of the rRNA in most mammalian cells. The standard Ribo-Zero Kit will remove cytoplasmic (nuclear-encoded) rRNA, but not mitochondrial rRNA.
7. How well do Ribo-Zero Kits perform with FFPE samples?
The Ribo-Zero Kits work very well on FFPE RNA. The design and configuration of the probes allows for the capture of very small pieces of rRNA, leaving the mRNA and other nonribosomal species in the supernatant.
8. Do Ribo-Zero Kits only remove rRNA?
Yes. While there is a possibility of some cross-hybridization between the capture probes and minute portions of other RNA species, the hybridization conditions are optimized for rRNA capture while minimizing undesirable cross-hybridization.
9. Why is the Ribo-Zero rRNA depletion method better for mRNA enrichment compared to poly(A) enrichment techniques, especially for degraded/FFPE samples?
Poly(A) enrichment involves the capture of the 3´-end of polyadenylated RNA, typically using oligo(dT) columns. If the RNA to be enriched is of high quality (RIN >9), then most of the mRNA will be recovered. However, if the input RNA is of poor quality (RIN <6), then the majority of the mRNA sequences will be lost and the resulting mRNA-Seq libraries will contain only 3´ sequences of the mRNA. By using the Ribo-Zero procedure, capture of the rRNA (intact or degraded) does not affect the mRNA sequences in the original RNA preparation. Libraries prepared after Ribo-Zero treatment therefore exhibit more even coverage density, and greatly reduced 3´ end bias compared to poly(A) enrichment. R-Z treatment also recovers non-poly(A), non-rRNA such as lincRNA that Poly(A) enrichment loses.
10. After I used a Ribo-Zero Kit, I noticed a peak at 100-140 nt in my Bioanalyzer traces (but no 16S/23S rRNA). Why is that?
This is normal and expected. The peaks observed in the 100- to 140-nt region on a Bioanalyzer trace correspond to tRNA, small noncoding RNAs, and small amounts of 5S rRNA (in cases where 5S rRNA removal is less efficient). These small RNAs are available for inclusion in a sequencing library if desired, but can be readily removed by gel or other size-selection techniques that are designed to remove small RNAs, either before or after library synthesis.
11. Can any of the Ribo-Zero Kits be used with environmental microbial samples?
Yes. The Ribo-Zero Bacteria Kit contains capture probes for both Gram-positive and Gram-negative bacteria, thus allowing removal of rRNA from an RNA sample that contains transcripts from many bacterial species.
12. What is the best way to quantify a rRNA-depleted sample?
Currently the best (and most valid) means of quantifying RNA recovered from a Ribo-Zero reaction is to use a fluorimeter and the RNA-binding dye Ribo-Green® (Life Technologies). Quantification by fluorimetry is fast and easy. If desired, results can be confirmed by qRT-PCR, selecting primer sets that recognize housekeeping genes or other readily recognizable amplicons. NanoDrop® spectrophotometers are not recommended due to their poor sensitivity for low mass/concentrations seen in a typical rRNA-depleted sample. Bioanalyzer-based quantification is not recommended either, as this method requires highly accurate and consistent pipetting of the size-ladder standard.