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 Poly(A) RNA Selection Kit V1.5 features new oligo(dT) Magnetic Beads (MB) compared to the V1.0 kit. The V1.5 kit protocol has been carefully optimized to maximize poly(A) RNA recovery. Additional specific changes made in the V1.5 Kit and protocol include:

• Updated Hybridization (HYB) and Bead Wash (BW) buffer compositions (for compatibility with new oligo(dT) Magnetic Beads).
• Reduction of the Magnetic Bead volume to 2 µl (vs. 10 µl in V1.0), which can be used for total RNA inputs up to 5 µg.

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.

Inputs higher than 5 µg of total RNA have not been directly tested. However, increasing the proportional bead volume to suit the input RNA amount will enable poly(A) selection from higher inputs.

In principle, use 2 µl of Magnetic Beads (MB) per 5 µg of total RNA. For input amounts that are not multiples of 5 µg, use next highest multiple of 5 to calculate the volume of MB (e.g., If using 32 µg of total RNA as input, calculate based on a 35 µg input: (35/5) = 7 x 2 µl = 14 µl of MB to add.
In this way the beads are always in slight excess for in-between input amounts, to ensure maximal poly(A) RNA recovery.

The table below gives recommendations for Bead Wash (BW) and Hybridization Buffer (HYB) volumes to use for scaled up reactions.