TITLE: NEBNext® Poly(A) mRNA Magnetic Isolation Module NEB #E7490S/L (Standard Protocol)
AUTHORS: Isabel Gautreau

[DESCRIPTION]
The NEBNext Poly(A) mRNA Magnetic Isolation Module is designed to isolate intact poly(A)+ RNA from previously isolated total RNA. The technology is based on the coupling of Oligo d(T)25 to 1 μm paramagnetic beads which is then used as the solid support for the direct binding of poly(A)+ RNA. Thus, the procedure permits the manual processing of multiple samples and can be adapted for automated high-throughput applications. Additionally, magnetic separation technology permits elution of intact mRNA in small volumes eliminating the need for precipitating the poly(A)+ transcripts in the eluent. Intact poly(A)+ RNA which is fully representative of the mRNA population of the original sample can be obtained in less than one hour. The new protocol for poly(A) mRNA enrichment (Section 1, Express Protocol) enables comparable performance to our current protocol (Section 2, Standard Protocol) using a substantially faster and more streamlined workflow.

[BEFORE_START]
Note: When using NEBNext Poly(A) mRNA Magnetic Isolation Module E7490 with NEBNext library preparation kits, follow the protocol in the respective library prep kit manual or see Appendix A. Modifications to elution buffer and volumes will need to be made at Step 40  to make the enriched mRNA directly compatible with the different library preparation workflows.

Note: For best results keep all the reagents used during the Poly(A) isolation except the NEBNext Oligo d(T)25 beads, on ice when not in use.

The protocol has been optimized using high quality Universal Human Reference Total RNA.

RNA Integrity

Assess the quality of the Input RNA by running the RNA sample on an Agilent Bioanalyzer RNA 6000 Nano/Pico Chip. For Poly(A) mRNA enrichment, high quality RNA with a RIN score > 7 is required.

RNA Sample Requirements

The RNA sample should be free of salts (e.g., Mg2+, or guanidinium salts, divalent cation chelating agents (e.g., EDTA or EGTA) or organics (e.g., phenol or ethanol). RNA must be free of DNA. gDNA is a common contaminant from RNA preps. It may be carried over from the interphase of organic extractions or when the silica matrix of solid phase RNA purification methods is overloaded. If the total RNA sample may contain gDNA contamination, treat the sample with DNase I to remove all traces of DNA (DNase is not provided in this kit). After treatment with DNase I the enzyme should be removed from the sample. Any residual activity of the DNase I may degrade the oligos necessary for the enrichment. DNase I can be removed from the extraction using phenol/chloroform extraction and ethanol precipitation.


[GUIDELINES]
Please refer to the product page on NEB.com for FAQs about this product.

Where larger volumes, customized or bulk packaging are required, we encourage consultation with the Customized Solutions team at NEB.
Please complete the NEB Custom Contact Form at www.neb.com/CustomContactForm to learn more.

Application

Isolation of poly(A)+ RNA transcripts from Total RNA for RNA library preparation and sequencing.

[STEPS]
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
1. 
 
Dilute the total RNA with nuclease-free water to a final volume of 50 µL in a nuclease-free 0.2 ml PCR tube and keep on ice.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
2. To wash the NEBNext Oligo d(T)25 Beads, add the following to a 1.5 ml nuclease-free tube. If preparing multiple libraries, beads for up to 10 samples can be added to a single 1.5 ml tube for subsequent washes (use magnet NEB #S1506 for 1.5 ml tubes). The purpose of this step is to bring the beads from the storage buffer into the binding buffer. The NEBNext RNA Binding Buffer (2X) does not have to be diluted for this step.

 

SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
3. Wash the beads by pipetting up and down 6 times.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
4. Place the tube on the magnetic rack at Room temperature for 2 min or until the solution is clear.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
5. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
6. Remove the tube from the magnetic rack.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
7. Add 100 µL NEBNext RNA Binding Buffer (2X) to the beads and wash by pipetting up and down 6 times. If preparing multiple libraries, add 100 µL RNA Binding Buffer per sample. The Binding Buffer does not have to be diluted.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
8. Place the tubes on the magnet and incubate at Room temperature until the solution is clear (~ 2 minutes).
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
9. Remove and discard the supernatant from the tube. Take care not to disturb the beads.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
10. Remove tubes from magnet and add 50 µL NEBNext RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous. If preparing multiple libraries, add 50 µL RNA Binding Buffer per sample. The Binding Buffer does not have to be diluted.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
11. Add 50 µL beads to each RNA sample from Step 1. Mix thoroughly by pipetting up and down 6 times. This first binding step removes most of the non target RNA.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
12. Place the tubes on the thermal cycler and heat the sample at 65 °C for 5 min and cool to 4 °C with the heated lid set at ≥ 75 °C to denature the RNA and facilitate binding of the mRNA to the beads.

SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
13. Remove tubes from the thermal cycler when the temperature reaches 4 °C.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
14. Resuspend the beads. Pipette up and down slowly 6 times to mix thoroughly.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
15. Place the tubes on the bench and incubate at Room temperature for 5 min to allow the RNA to bind to the beads.

SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
16. Resuspend the beads. Pipette up and down slowly 6 times to mix thoroughly.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
17. Incubate for 5 more minutes on the bench at Room temperature to allow the RNA to bind to the beads.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
18. Place the tubes on the magnetic rack at Room temperature for 2 min or until the solution is clear to separate the poly(A) RNA bound to the beads from the solution.

SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
19. Remove and discard all of the supernatant. Take care not to disturb the beads.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
20. Remove the tubes from the magnetic rack.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
21. Wash the beads by adding 200 µL of NEBNext Wash Buffer to remove unbound RNA. Pipette the entire volume up and down 6 times to mix thoroughly.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
22. Place the tubes on the magnetic rack at Room temperature for 2 min or until the solution is clear.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
23. Remove and discard all the supernatant from each well of the tube. Take care not to disturb the beads.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
24. Remove the tubes from the magnetic rack.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
25. Repeat Steps 21–24
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
26. Add 50 µL of NEBNext Tris Buffer (provided in NEB #E7490 kit) to each tube. Gently pipette the entire volume up and down 6 times to mix thoroughly.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
27. Place the tubes on the thermal cycler. Close the lid and heat the sample at 80 °C for 2 min, then cool to 25 °C with the heated lid set at ≥ 90 °C to do the first elution of the mRNA from the beads.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
28. Remove the tubes from the thermal cycler when the temperature reaches 25 °C.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
29. Add 50 µL of NEBNext RNA Binding Buffer (2X) to each sample to allow the RNA to re-bind to the same beads. The Binding Buffer does not have to be diluted. Gently pipette the entire volume up and down 6 times to mix thoroughly.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
30. Incubate the tubes on the bench at Room temperature for 5 min.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
31. Resuspend the beads. Pipette up and down slowly 6 times to mix thoroughly.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
32. Incubate the tubes on the bench at Room temperature for 5 more minutes to allow the RNA to bind to the beads.

SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
33. Place the tubes on the magnetic stand at Room temperature for 2 min or until the solution is clear.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
34. Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
35. Remove the tubes from the magnetic rack.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
36. Wash the beads once with 200 µL of NEBNext Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly. Spin down the tube briefly to collect the liquid from the wall and lid of the tube.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
37. Place the tubes on the magnetic rack at Room temperature for 2 min or until the solution is clear.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
38. Remove and discard all of the supernatant from each tube. Take care not to disturb the beads.
 

SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
39. Remove the tubes from the magnetic rack.
 

SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
40. Elute the mRNA from the beads by adding 17 µL of the NEBNext Tris Buffer, mix by pipetting 6 times and incubate the samples at 80 °C for 2 min, then cool to 25 °C. Immediately, place the tubes on the magnetic rack for 2 min or until the solution is clear.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
41. Collect the purified mRNA by transferring 15 µL of the supernatant to a clean nuclease-free PCR Tube. Place on ice if using immediately. Alternatively, the sample can be placed at -80 °C for long term storage.
SECTION: Starting Material: 1–5 μg* of DNA-free total RNA (Standard Protocol)
42. Assess the Yield and the Size Distribution of the purified mRNA. Run 1 µL on the Bioanalyzer using a RNA Pico Chip or the Tapestation using a High Sensitivity RNA ScreenTape. You may have to dilute your sample before loading.