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.
are aliquoted and washed.
are hybridized to oligodT beads.
and tRNA,will not be captured by the oligodT beads
and will be washed away.
by heating in water.
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 email@example.com.
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.
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.
|Total RNA Input*||Step 1
Volume of MB
|Step 3 / 4
Volume of BW
| Step 5
HYB added to MB
RNA in Volume
|Step 10 / 11
Volume of BW
|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.
User Guide – update 11.11.2020
Material Safety Datasheets
If you need more information about our products, please contact us through firstname.lastname@example.org or directly under +43 1 345 1212-41.