Alpha and beta globin mRNA depletion from total RNA extracted from human blood
Oxford Nanopore
Alpha and beta globin mRNA depletion from total RNA extracted from human blood
FOR RESEARCH USE ONLY
Contents
Introduction
Materials
Methods
Results
Sequencing performance
Change log
Introduction
This protocol describes a method to perform globin depletion on a total RNA sample extracted from human blood. Prior to globin depletion, the blood was collected and stabilized in PAXgene® Blood RNA Tubes and extracted with PAXgene® Blood RNA Kit. The extracted RNA was globin depleted using GLOBINclear™-Human Kit. Sequencing performance was assessed using the PCR-cDNA Sequencing Kit.
Materials
- Total RNA extracted from human blood
- GLOBINclear™-Human Kit
- Ethanol
- Isopropanol
- Vortex
- 1.5 ml Eppendorf tubes
- Incubator with capacity for 50°C and 58°C
- Magnetic rack
Optional (if RNA concentration ≤70 ng/µl)
- Glycogen
- 5 M ammonium acetate
- -80°C freezer
- Refrigerated mini-centrifuge with capacity for 12000 x g
- TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
Methods
If the total RNA concentration is below 70 ng/µl continue to step 2, otherwise continue to step 10.
Transfer an adequate volume of the total RNA sample (ideally close to 10 µg) to a 1.5 ml Eppendorf tube and add 0.1 volume of 5 M ammonium acetate, 5 µg glycogen, and 3 volumes of ethanol.
Invert 10 times and place the mixture at -80°C for 30 minutes.
Centrifuge at 12000 x g for 30 minutes at 4°C
Discard the supernatant and add 1 ml of ice cold 70% ethanol; invert the tube to mix.
Centrifuge at 12000 x g for 10 minutes at 4°C.
Discard supernatant.
Repeat steps 5 to 7.
Dissolve the RNA pellet in 15 µl of TE and use 1 µl to assess concentration.
Follow the recommended protocol (steps B1 to F7, pages 7-12 of the handbook).
Results
- Yield: ~50-90% of input
- OD 260/280: 2.03
- OD 260/230: 0.48 (this low value could be due to carry-over of reagents or magnetic beads during the globin depletion process).
- Agilent Bioanalyzer RNA 6000 Nano Kit, RIN: 9.2
Sequencing performance
Libraries for nanopore sequencing were prepared from 50 ng of both total RNA and globin depleted RNA, using the PCR cDNA Sequencing Kit:
- Read length profile:
Figure 1. The length distribution of reads from total RNA and globin depleted RNA from human blood. The read length distribution from total RNA is dominated by a group of alpha and beta globin transcripts around 800 bp in length. The globin depletion effectively removes the bulk of these transcripts.
Change log
| Version | Change |
|---|---|
| v1, June 2019 | Initial protocol publication |