Description

Poly(A) RNA Selection Kit V1.5

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

Optimized V1.5 Upgrade

The upgrade V1.5 contains new oligo(dT) Magnetic Beads (MB), compared to the V1.0 kit. The beads and solutions included in the V1.5 kit offer improved performance.

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.

Poly(A) Figure 1

Figure 1. Bioanalyzer traces of Universal Human Reference Total RNA (UHRR, red trace) and poly(A) selected RNA from 5 µg UHRR (blue trace).

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.

Rapid Turnaround

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

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 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.

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.

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.

Total RNA Input* Step 1
Volume of MB
Step 3 / 4
Volume of BW
 Step 5
HYB added to MB
Step 6
RNA in Volume
Step 10 / 11
Volume of BW
Step 12
Elution Volume
100 µg 20 µl ≥200 µl 100 µl HYB 100 µl ≥200 µl 50 µl
50 µg 10 µl ≥200 µl 50 µl HYB 50 µl ≥200 µl 25 µl

*Input amounts are for exemplary purposes only.

Downloads

pdf  User Guide – update 11.11.2020

Material Safety Datasheets

MSDS Information can be found in the Documents page.

If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.

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