6A. Quality Control Check and Size Selection using 6% Polyacrylamide Gel  NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)

This protocol follows Protocol for use with NEBNext Small RNA Library Prep Set for Illumina E7300E7580E7560E7335

6A.1. Purify the PCR amplifed cDNA construct (100 µl) using a Monarch PCR & DNA Kit.

IMPORTANT: Use the 7:1 ratio of binding buffer: sample. Discard the flow through after each centrifugation step.

6A.2. Elute amplifed DNA in 27.5 µl Nuclease-free Water.

 Safe Stopping Point: It is safe to store the library at -20°C.

6A.3. Load 1 µl of the purifed PCR reaction on the Bioanalyzer using a DNA 1000 chip according to the manufacturer's instructions (Figure 1).

Figure 1: Typical results from (A) human brain and (B) rat testis total RNA libraries before size selection.
The 143 and 153 bp bands correspond to miRNAs and piRNAs, respectively. The bands on the Bionalyzer electropherograms resolve in sizes ~ 6-8 nucleotides larger than sizes observed on PAGE gels and can shift from sample to sample due to an incorrect identifcation of the marker by the bioanalyzer software. miRNA peak should be ~ 143-146 bp 

6A.4. Mix the purifed PCR product (25 µl) with 5 µl of Gel Loading Dye, Blue (6X).

Note: Vortex the Gel Loading Dye, Blue throughly to mix well before using.

6A.5. Load 5 µl of Quick-Load pBR322 DNA-MspI Digest in one well on the 6% PAGE 10-well gel.

6A.6. Load two wells with 15 µl each of mixed amplifed cDNA construct and loading dye on the 6% PAGE 10-well gel.

6A.7. Run the gel for 1 hour at 120 V or until the blue dye reaches the bottom of the gel. Do not let the blue dye exit the gel.

6A.8. Remove the gel from the apparatus and stain the gel with SYBR Gold nucleic acid gel stain in a clean container for 2–3 minutes and view the gel on a UV transiluminator (Figure 2).

Figure 2: Shows typical results from Human Brain (A) and Rat Testis (B) Total RNA libraries. The 140 and 150 bp bands correspond to miRNAs (21 nt) and piRNAs (30 nt), respectively.

6A.9. The 140 and 150 nucleotide bands correspond to adapter-ligated constructs derived from the 21 and 30 nucleotide RNA fragments, respectively. For miRNAs, isolate the bands corresponding to ~140 bp. For piRNAs, isolate the band corresponding to ~150 bp. For other small RNA, the band size may be different.

6A.10. Place the two gel slices from the same sample in one 1.5 ml tube and crush the gel slices with the RNase-free Disposable Pellet Pestles and then soak in 250 µl DNA Gel Elution buffer (1X).

6A.11. Rotate end-to-end for at least 2 hours at room temperature.

6A.12. Transfer the eluate and the gel debris to the top of a gel fltration column.

6A.13. Centrifuge the filter for 2 min at > 13,200 rpm.

6A.14. Recover eluate and add 1 µl Linear Acrylamide, 25 µl 3M sodium acetate, pH 5.5 and 750 µl of 100% ethanol.

6A.15. Vortex well.

6A.16. Precipitate in a dry ice/methanol bath or at –80°C for at least 30 minutes.

6A.17. Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.

6A.18. Remove the supernatant taking care not to disturb the pellet.

6A.19. Wash the pellet with 80% ethanol by vortexing vigorously.

6A.20. Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.

6A.21. Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.

6A.22. Resuspend pellet in 12 µl TE Buffer.

6A.23. Load 1 µl of the size selected purifed library on a 2100 Bioanalyzer using a DNA 1000 or High Sensitivity DNA chip according to the manufacturer's instructions (Figure 3).

6A.24. Check the size, purity, and concentration of the sample.

Figure 3: Electropherogram trace of the gel size selected purified library from human brain total RNA.