Figure 1.
Workflow illustrating the procedures for Organism-Specific Probe Selection and rRNA depletion.
A. Workflow of Organism-Specific Probe Selection (OSPS) program. OSPS was used to screen for unique sequences of 16S rRNAs and 23S rRNAs that have no significant similarity to other transcripts in the same organism. B. Overall procedures of rRNA depletion. Sequences for probes were amplified and cloned into an in-house pT1 system, and the RNA probes were which by in vitro transcribed with biotinylated UTP and tested for rRNA depletion efficiency. The best probes were selected and combined for further rRNA depletion.
Figure 2.
Evaluation of the removal efficiency of rRNA from total RNA using probes designed by OSPS.
The rRNA levels were measured before and after rRNA depletion by real-time PCR. Untreated sample was included for comparison and MSMEG_5072 gene was used as a normalization gene. * means p-value <0.001. A. Depletion efficiency for 16S rRNA. Five probes of 16S rRNAs were used for depletion separately. B. Depletion efficiency for 23S rRNA. Four probes of 23S rRNAs were used to deplete 23S rRNA. C. Depletion of rRNAs from total RNA of Mycobacterium smegmatis by combined probes. Total RNA before and after depletion were loaded and separated on an agarose gel. Lane 1, 1.5 µg RNA marker; lane 2, RNA sample was depleted using the MICROBExpress™ Bacterial mRNA Enrichment Kit (Ambion); lane 3, RNA sample was depleted by combined probes (16S-3 and 23S-1); lane 4, 0.5 µg of untreated RNA.
Figure 3.
Integrity of mRNA after rRNA depletion was determined by real-time PCR.
The expression level of rRNA and 14 genes of different abundance were measured before and after rRNA depletion using MSMEG_5072 gene as a housekeeping control. Abundance of selected genes and the gene ID were shown. A. Relative fold change of selected mRNA and rRNA after depletion using MICROBExpress™ Bacterial mRNA Enrichment. B. Fold change of selected mRNA and rRNA after depletion using probes designed by OSPS.