Description

Poly(A) RNA Selection Kit

The Poly(A) RNA Selection Module enables the rapid and highly specific enrichment of polyadenylated RNAs from total RNA samples.

Rapid Turnaround

Polyadenylated RNAs can be isolated from total RNA samples within about one hour.

Various Downstream Applications

Isolated mRNA can directly be used for RNA-Seq library preparation (e.g., SENSE Total RNA-Seq kit), SAGE, CAGE, cloning, microarrays, cDNA synthesis, and others.

Highly Specific for Poly(A) RNA

Other RNA species (rRNA and tRNA) do not contain poly(A) sequences and therefore will not bind to the oligo(dT) beads.
PolyA_Graph

Figure 1. Bioanalyzer traces of the poly(A) RNA enrichment using 5 µg of Universal Human Reference RNA (UHRR) (A) input with the Poly(A) RNA Selection Kit. Recovery of poly(A) selected UHRR (B) is about 2 % of the initial input amount.

Workflow

Preparation of Beads and Denaturation of RNA
Step 1:
The total RNA Is briefly denatured and magnetic beads
are aliquoted and washed.
01polya_workflow
Hybridization of poly(A) RNA
Step 2:
The polyadenylated 3’ ends present in most mRNAs
are hybridized to oligodT beads.
02polya_workflow02progressbar_polyA
Poly(A) RNA Enrichment
Step 3:
Any RNA without poly(A) stretches, such as rRNA
and tRNA,will not be captured by the oligodT beads
and will be washed away.
03polya_workflow
Poly(A) RNA Elution
04polya_workflow04progressbar_polyA
Step 4:
The poly(A) RNA is eluted from the olidodT beads
by heating in water.

FAQ

Frequently Asked Questions

Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.

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.

PolyA_FAQ_Bioanalyzer-A

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.

PolyA_FAQ_Bioanalyzer-B

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.

Downloads

pdf  User Guide – update 07.06.2019
pdf  Product Flyer

Material Safety Datasheets

pdf  MSDS information for Poly(A) RNA Selection Kit

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