The oligodT consists of 30 Ts.

The poly(A) RNA Selection Module enriches for all RNAs that have a poly(A) tail, such as mRNA, but also mitochondrial rRNA, which can make up to 4 % of the isolated poly(A) RNA (=0.08% of the total RNA). rRNA removal was evaluated by an RNA-Seq experiment using a mRNA-Seq protocol (SENSE mRNA-Seq) and only 0.00015 % of all reads were mapping to rRNA.

The SENSE mRNA-Seq kit already includes the oligodT beads in the kit and doesn´t need to be bought separately. For the SENSE Total RNA-Seq kit the Poly(A) RNA Selection Kit can be used for an efficient poly(A) isolation. If an mRNA-Seq experiment is planned we recommend using the SENSE mRNA-Seq kit as here the total RNA input can be significantly lower as the poly(A) RNA does not need to be eluted form the beads plus the SENSE mRNA-Seq kit offers more flexibility regarding the library size selection.

Repeated rounds of poly(A) selection are not required due to the high specificity of the poly(A) selection. One poly(A) selection round is enough. Repeated rounds are often performed to remove unspecifically bound rRNA.

The amount of poly(A) RNA varies depending on the tissue and cell type the total RNA is derived from. Poly(A) RNA usually makes up 1-3 % of the total RNA.

Agilent’s Bioanalyzer and other electrophoresis platforms often try to detect 18S and 28S rRNA peaks at all costs. Therefore, highly abundant transcripts are sometimes wrongly identified as rRNA.

In the traces below you’ll see that the Bioanalyzer software classifies two peaks within the trace of a poly(A) selected sample as 18S and 28S rRNA (Figure A). However, the overlay with diluted total RNA clearly demonstrates that those peaks are not rRNA peaks (Figure B). Poly(A) selection was successful and no rRNA was left. This was also confirmed by NGS sequencing.

Figure A. Bioanalyzer trace after the Poly(A) RNA Selection using 5 µg of Universal Human Reference RNA (UHRR). The Bioanalyzer software wrongly classifies two peaks within the trace of the poly(A) selected sample as 18S and 28S rRNA.

Figure B. Overlay of Bioanalyzer traces before and after the Poly(A) RNA Selection. The overlay with diluted total RNA clearly demonstrates that those peaks identified as rRNA (in A) are not rRNA peaks.
Red trace: diluted total Universal Human Reference RNA (UHRR), intact (RIN 8.5). Blue trace: poly(A) RNA selected RNA.