Introduction
When working with RNA extracted from complex biological samples—such as tissues, blood, or cell cultures—one common challenge is the presence of highly abundant, non-target RNA. These dominant RNA species, like globin mRNA and ribosomal RNA, can easily overshadow the transcripts you're actually interested in. This is especially relevant in studies involving blood-stage parasites like Plasmodium, where parasite-derived mRNAs are often low in abundance compared to the host RNA background. Other relevant scenarios include studies where a less abundant organism or cell type is present. Without proper RNA enrichment, the signals from these low-abundance transcripts may be lost.
To address this, researchers typically use one of two strategies to enrich for protein coding or target-related RNA:
- Enrich for target mRNA using methods that selectively capture polyadenylated transcripts (if applicable), or
- Deplete host globin and ribosomal RNAs using targeted probes or kits.
NEBNext Globin & rRNA Depletion Protocol In Brief:
- 200ul of PCR tubes
- 80% freshly prepared ethanol (10ml 1ml Ethanol + 9ml of nuclease-free water).
- RNA XP cleanup beads
- 0.5-1ug of total RNA as input
- Magnetic Rack
- Esky of ice
1) HYBRIDISATION (on ice)
5ul of total RNA (0.5-1ug)
3ul of NEBNext Globin & rRNA depletion solution
2ul Hybridization mix
5ul nuclease-free water
Thermal cycler setting: 95oC for 2min (with heated lid set to 105oC; Ramp down to 22oC at 0.1oC/sec for 5min; Hold at 4oC
Briefly spin
2) RNase H DIGESTION (on ice)
15ul Hybridized RNA from step above
2ul NEBNext Thermostable RNase H
2ul RNase H Reaction Buffer
1ul Nuclease-free water
total vol. is 20ul
Pipette up-and-down x10 times
Briefly spin down contents
Incubate in pre-heated thermal cycler
Thermal cycler setting: 50oC for 30min (with heated lid set to 55oC; Hold at 4oC
Briefly spin down contents
3) DNase I DIGESTION (on ice)
(tip: make a mastermix of the reagents if you have more than 1 sample)
20ul RNase H treated RNA
5ul DNase I Reaction Buffer
2.5ul DNase I
22.5ul Nuclease-free water
Total vol. is 50ul
e.g. for x4 preps, mastermix
22ul of DNase I reaction Buffer
11ul DNase I
99ul Nuclease-free water
Aliquot 30ul of mastermix into individual tubes
Add 20ul of appropriate RNase H treated RNA
Mix up-and-down x10 times
Briefly spin
Incubate in pre-heated. Thermal cycler setting: 37oC for 30min (with lid set to 40oC or off).
Briefly spin.
4) Purification Using RNA XP Clean Up Beads
- Vortex beads
- 90ul of beads to RNA samples
- Pipette up-and-down x10 times
- Incubate tubes for 15min on ice
- Place on magnetic rack until beads collect on side
- Carefully remove and DISCARD supernatant
- 200ul of 80% ethanol (while on magnetic rack)
- Incubate for 30sec then remove supernatant & discard
- Repeat 80% ethanol wash
- Remove residual ethanol and air dry for 5min
- Add 7ul nculease-free water to lute RNA from beads
- Pipette up-and-down x10 times
- Briefly spin
- Incubate for 2min
- Place on magnet
- Aspirate 5ul of eluted RNA
Store at -20oC until needed
Watch our video on how mRNA enrichment is typically performed in the lab.
Both approaches help amplify the signal from the parasite and reduce the noise from the host, allowing for a clearer view of the parasite’s gene expression landscape—especially those subtle transcripts that might hold clues to its biology, drug resistance, or lifecycle.
Conclusion
Regardless of the system you're studying, enriching for target RNA or depleting abundant background RNA is a key step in generating meaningful transcriptomic data. By minimising unwanted host RNA, you can amplify the expression signals of interest—whether from pathogens, rare cell types, or low-expression genes—allowing deeper insights into the biology at play.
Subscribe by Email
Follow Updates Articles from This Blog via Email
No Comments