RNA extraction & depletion

Nils Weng; Ebba Perman

Dec 11, 2024

Version 1

RNA extraction & depletion V.1

DOI

dx.doi.org/10.17504/protocols.io.36wgq3y6klk5/v1

Nils Weng¹,
Ebba Perman¹

¹Swedish University of Agricultural Sciences

Nils och Ebba

Nils Weng

SLU

DOI: dx.doi.org/10.17504/protocols.io.36wgq3y6klk5/v1

Protocol Citation: Nils Weng, Ebba Perman 2024. RNA extraction & depletion . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3y6klk5/v1Version created by Nils Weng

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: November 21, 2023

Last Modified: December 11, 2024

Protocol Integer ID: 114469

Keywords: RNA extraction, RNA depletion

Abstract

This protocol provides a detailed methodology for the extraction and purification of RNA from both enrichment cultures and sampels from anaerobic digesters. The targeted microbial communities include organisms relevant to the anaerobic digestion process.

Protocol overview:

1. Sample preparation
Protocol includes description for; 
a) Samples from enrichment/pure cultures. 
b) Samples from anaerobic digesters.

2. RNA extraction & purification
RNA is extracted using TriZol/Chloroform extraction.
Extraction is followed by purification using Quick-RNA™ Fecal/Soil Microbe Microprep kit, excluding the cell lysis and extraction step included in the kit. Kit protocol is followed from step 4.

3. Quality control
Agilent 2100 BioAnalyzer used for quality control and concentration measurement.
The assay "Prokaryote Total RNA Nano Series II"  is used at this step.

4. rRNA depletion 
Ribosomal RNA is removed using the Pan-Prokaryote riboPOOL kit, followed by ethanol precipitation.

5. Concentration measurement
Agilent 2100 BioAnalyzer used for quality control and concentration measurement.
The assay "mRNA Nano Series II"  is used at this step.

It is recommended that Sample preparation, RNA extraction & purification, and Quality control is carried out without pause. After the quality control, samples can be stored at -80ºC until further processing. 

Materials

For materials needed for RNA depletion see section 25
-------------------------------------------------------------------
For each RNA extraction sample the following material is needed: 

Tubes/filters/columns
1x ZR BashingBead Lysis Tube
2x Zymo-Spin™ IIICG Column
1x Zymo-Spin™ III-HRC Filter
1x Zymo-Spin™ IC Column
5x Collection tubes (Could be reused at some steps)
3x 2.5ml RNAse free Eppendorf tubes

Solutions/Buffers:
1 ml       TriZol Reagent/750 μl TriZol LS
200 µl   Chloroform   
1-2 ml    Ethanol (95-100%)
800 µl    RNA Prep Buffer (kit)
115 µl    DNase/RNase-Free Water (kit)
600 µl    Prep Solution (kit)
200 µl    RNA Binding Buffer (kit)
400 µl    RNA Prep Buffer (kit)
1100 µl  RNA Wash Buffer (kit)

Enzymes/buffers for enzymes(DNA depletion)
DNase I and DNA Digestion Buffer Set (Zymo Research)
Stored in -20°C freezer

35 µl     DNA Digestion Buffer 
5 µl        DNase I (1U/μL)

-------------------------------------------------------------------

Equipment and Instruments used:
Bead-beater:  MP FastPrep-24 (MP Biomedicals)
Bioanalyzer: Agilent 2100 Bioanalyzer (Agilent Technologies)
Refrigerated Microcentrifuge: Centrifuge 5424 R (Eppendorf AG)
Heat block: Thermal Shake lite (VWR International)
Refrigerated Centrifuge for 50ml falcon tubes: Jouan CR322 (Thermo Fisher Scientific)

Protocol materials

ReagentZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)Zymo Research 
ReagentZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)Zymo Research 

Safety warnings

Warning: Trizol and chloroform are toxic and must be handled in a fume hood. Contaminated materials should be disposed of as hazardous waste in accordance with safety regulations. Due to the strong odor of contaminated materials, it is preferable to store them in a fume hood until disposal.

RNA extraction preparations

Preparations to do before starting the RNA extraction (in the morning of extraction):

Chill centrifuge to 4° C 
Chill ethanol, Trizol LS, and chloroform at -20oC. 
Clean workstation and equipment with ethanol and RNAse Away (or any other RNAse decontamination solution)
Get a box of ice to keep samples on

Sample Preparation

Sample Preparation

Sample extraction procedure differs slightly depending on sample type. 
For sample from pure culture or enrichment culture ⇒ do step 2.1-2.5
For sample from anaerobic digester ⇒ do step 2.6-2.8

Extraction from pure cultures/enrichment cultures

Note
The sample volume required to obtain sufficient RNA yield varies significantly depending on the sample type. The amounts provided here are based on mesophilic batch cultivation of syntrophic acid degrading enrichment cultures at the final stage of substrate degradation.

For each sample 3x50 ml were taken and put into 3 50ml falcon tubes while flushing with N2

# Tip: Try to be as quick as possible and keep samples on ice when possible.

Centrifuge in pre-cooled centrifuge at 4° C, 5500 RPM for 30 minutes

Centrifigation5500 rpm, 4°C, 00:30:00  

30m

Discard the supernatant by pouring/gently flicking the tube.
Put the pellets on ice.

#Tip, try to remove as much liquid as possible without losing the pellet.

Resuspend the pellet in 750 µl chilled Trizol LS, mix by pipette.
Transfer solution to the next 50ml falcon tube from the same sample, redo mixing and lastly transfer to the last 50ml falcon tube and mix througly.

Amount750 µL  Trizol LS  


Note
#Discussion
In the case of flocculating enrichment cultures, the final pellet volume is quite large after pooling. Therefore the more concentrated  Trizol LS is used which requires a lower ratio between the sample and Trizol LS as compared to normal Trizol.

The guideline is either 0.75ml Trizol LS or 1ml Trizol  for 0.25ml of Sample Volume. 

You also have to make sure make sure that the final volume should fit in the bead beating tube at the next step. The final volume of pellet + Trizol should be ≈ 1000µl.
The absolute maximum that is possible to add to the bead beating tube is ≈1400µl.

Transfer the mixed sample to a ZR BashingBead Lysis Tube

ReagentZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)Zymo Research  

Note
Continue at step 3.

Extraction from digestate samples (from anaerobic digestion process)

Sample the process at a specified time-point. 
Note
Ensure the workstation is clean and ready before sample collection. As RNA degrades quickly in the samples, try to work fast and immediately proceed to the next step. 

Add 200-250 μl of the sample to a ZR BashingBead Lysis Tube. 
Put tube on ice. 

ReagentZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)Zymo Research  

Note
Depending on the viscosity and particle size, samples can either be transferred by pipetting or scooping. Determine the volume using a scale.

Add 1 ml chilled TriZol Reagent (alt. 750 μL TriZol LS)

Amount1 mL TriZol Reagent  

Note
Continue at step 3.

RNA extraction

6m 30s

Homogenize samples using a bead-beater at speed 6.0 for 40 s

Duration00:00:40 Speed 6.0  

Note
Warning: Mark the side of the BashingBead Lysis Tube as the bead-beating will wear away any markings made on the top of the tube.

40s

Add 200 µl chilled chloroform to the cell mixture.
Mix well by inversion.

Amount200 µL Chloroform  

Centrifuge the mixture in pre-cooled centrifuge at 4° C, 14000 rcf for 10 minutes.

Centrifigation14000 rcf, 4°C, 00:10:00  

10m

(The following steps follow the Quick-RNA™ Fecal/Soil Microbe Microprep protocol, starting from step 4.)

#### OBS##### 
All centrifugation for RNA extraction should be done at 10,000 -16,000 rcf at room temperature unless stated otherwise.


Phase separation after centrifugation. Example of sample from enrichment culture.
Note: For samples from digesters, the aqueous phase will have a brown color.
Transfer 400 µl (or as much as available) of the aqueous phase into an RNase-free tube (not provided) and add 1 volume of  RNA binding buffer. Mix well.

AmountX(400) µL Supernatant  
AmountX(400) µL RNA binding Buffer  
 
#Tip: If more than 400 µl can be extracted do that, but you will need to repeat step 7 in order to filter all of the liquid.

Transfer up to 800 μl of the mixture into a  Zymo-Spin™ IIICG Column in a clean 2.5 ml Eppendorf tube and centrifuge at 5,000 x g for 60 seconds. Save the flow-through!

Centrifigation5000 rcf, 00:01:00  

#Tip: Column might need to be loaded multiple times depending on sample volume. 

To the flow-through, add an equal volume ethanol (95-100%) (1:1) and mix well.
Amount1 µL 95-100% Ethanol  

#Tip: A 2.5ml Eppendorf tube may be too small if the supernatant volume was large. In this case just transfer to RNase free 5ml Eppendorf tube or divide the volume in half and use two 2.5ml tubes.

Transfer the mixture into a new Zymo-Spin™ IIICG Column in a Collection Tube and centrifuge. Discard the flow-through.

Centrifigation10000-16000 rcf, 00:00:30  

#Column might need to be loaded multiple times depending on sample volume. Just repeat this step in that case.

30s

Add 400 µl RNA Prep Buffer to the column and centrifuge fo 30s. Discard the flow-through. 

Amount400 µL RNA Prep Buffer  
Centrifigation10000-16000 rcf, 00:00:30  

30s

Transfer the column into a nuclease-free tube (not provided).
Add 100 µl DNase/RNase-Free Water directly to the column matrix and centrifuge for 2 minutes at 5000 rcf.

Amount100 µL DNase/RNase-Free Water   
Centrifigation5000 rcf, 00:02:00  

Zymo-Spin™ III-HRC Filter preparation:

Place a Zymo-Spin™ III-HRC Filter in a new Collection Tube and add 600 µl Prep Solution. Centrifuge at 8,000 x g for 4 minutes.

Amount600 µL Prep Solution  
Centrifigation8000 rcf, 00:03:00  

Transfer the eluted RNA (step 11) into the prepared filter in a nuclease-free tube (not provided) and centrifuge at 16,000 x g for 4 minutes.

Amount100 µL Eluted RNA  
Centrifigation16000 rcf, 00:04:00  

Add 200 µl RNA Binding Buffer to the filtered RNA (step 13) (2:1) and mix well.

Amount200 µL RNA Binding Buffer  

Add an equal volume ethanol (95-100%) (1:1) and mix well.

Amount300 µL 100% ethanol  

Transfer the mixture into a new Zymo-Spin™ IC Column in a Collection Tube and centrifuge. Discard the flow-through.

Centrifigation10000-16000 rcf, 00:00:30  

Note
IMPORTANT: At this point, when the RNA has bound to the IC Column, DNA can be removed using DNase I treatment.  See 16.1 - 16.3

30s

In-column DNase I treatment

6m 30s

Following RNA binding step (step 16), add 400 µl RNA Wash Buffer to the column, centrifuge and discard the flow-through.

Amount400 µL RNA Wash Buffer  
Centrifigation10000-16000 rcf, 00:00:30  

30s

For each sample to be treated, prepare DNase I Reaction Mix (see table below) in an RNase-free tube (not provided) and mix by gentle inversion.

For each sample:
35 µl DNA Digestion Buffer
5 µl DNase I

Add 40µl of the DNase I Reaction Mix to the column matrix and incubate at room temperature (20-30°C) for 15 minutes. Proceed with the purification (Step 17)

Amount40 µL DNase I reaction mix  
Duration00:15:00   
TemperatureRoom temperature   

TIP: If you are quantifying RNA concentration using Bioanalyzer take out the kit to let it come up to room temperature at this point.

15m

RNA extraction

6m 30s

Add 400 µl RNA Prep Buffer to the column and centrifuge. Discard the flow-through.

Amount400 µL RNA Prep Buffer  
Centrifigation10000-16000 rcf, 00:00:30  

30s

Add 700 µl RNA Wash Buffer to the column and centrifuge. Discard the flow-through.

Amount700 µL RNA Wash Buffer  
Centrifigation10000-16000 rcf, 00:00:30  

30s

Add 400 µl RNA Wash Buffer and centrifuge the column for 1 minute to ensure complete removal of the wash buffer. Then carefully, transfer the column into a nuclease-free tube (not provided).

Amount400 µL RNA Wash Buffer  
Centrifigation10000-16000 rcf, 00:01:00  

#Tip: The centrifugation step can be repeated without addition of Wash Buffer to ensure that all Wash Buffer is removed.

Add 15 µl DNase/RNase-Free Water directly to the column matrix, incubate for 2 minutes and centrifuge.

Amount15 µL DNase/RNase-Free Water  (Amount can be varied)   
Duration00:02:00 Incubation  
Centrifigation10000-16000 rcf, 00:00:30  

Alternatively, for highly concentrated RNA use ≥ 6 µl elution. 
The eluted RNA can be used immediately for QC control or stored frozen.


The RNA can now be frozen in  the -80 ° freezer.  Its recommended to do a QC control using Bioanalyzer at this point to avoid repeated freeze/thawing of the RNA samples. Alternatively you can stop here and aliquot 2µl of each sample, freeze everything and continue to do a quality check at a later stage.

2m 30s

Quality Control - BioAnalyzer

Info: For QC control the BioAnalyzer was used in combination with the Agilent RNA 6000 Nano Kit. 
The protocol followed can be found here (Protocol).

For each sample to be analyzed 1 µl of eluted RNA is needed.

Amount1 µL RNA sample  

Check whether there is fresh filtered Gel prepared or if new gel has to be prepared (Lasts 4 weeks)

Decontaminate the Electrodes:

Slowly fill one of the wells of an electrode cleaner with 350 µl RNaseZAP. 
 Open the lid and place the electrode cleaner in the Agilent 2100 Bioanalyzer. 
Close the lid and leave it closed for about 1 minute. 
Open the lid and remove the electrode cleaner. Label the electrode cleaner and keep it for future use. You can reuse the electrode cleaner for all 25 chips in the kit. 
Slowly fill one of the wells of another electrode cleaner with 350 µl RNase-free water.
Place the water-filled chip in the Agilent 2100 bioanalyzer, close the lid and leave for 10s.
Remove the chip and leave the lid open for 10s to dry the electrode. 

Run the analysis according to manufacturers instructions.
Use the assay "Prokaryote Total RNA Nano Series II"

Depletion of ribosomal RNA

10m

rRNA depletion is performed with the riboPOOL Kit. For more detailed information read the protocol that comes with the kit. 
The protocol presented here is based on the kit protocol.
Newest protocols found here:
https://www.sitoolsbiotech.com/protocols.php

The current protocol is based on the following:
https://www.sitoolsbiotech.com/pdf/riboPOOLkitManualv1-6_2023.pdf

Reagents and storage requirements for RNA depletion

Stored in freezer:
LA       -    Linear acrylamide 
RP       -    riboPOOL lyophilized,(aliquoted by user)
RNAse inhbitor    RiboLock (40u/µl)

Stored in fridge :
SMB    -    Streptavidin-coated magnetic beads (SMB)

Stored in room temperature:
DB    -    Depletion buffer
HB    -    Hybridization buffer
SA    -    Sodium acetate, 3M
H2O    -    Nuclease-free water

Reagents that has to be prepared in advance:
BRB - Bead Resuspension Buffer
            0,1 M NaOH
            0.05M NaCl

BWB - Bead Wash Buffer
            0.1M NaCl

Hybridization of riboPOOL to rRNA

To 14 μl of RNA sample (containing 100 ng - 5 μg of total RNA), add: 
If sample volume is > 14 μl, adjust HB volume accordingly to 0.25X total volume. Total volume however should not exceed 40 μl. 

Amount1 µL resuspended RP  
Amount5 µL Hybridization Buffer (HB)  
AmountX µL RNase inhibitor (RiboLock)  


#Tip:
For RNase inhibitor the final concentration should be 1 U/μl. Given a RiboLock concentration of 40 U/μl and a working volume of 20 μl (14+1+5) -> 0.5μl of RiboLock should be added to each sample

Vortex and spin down droplets

Incubate at 68°C for 10 min to denature RNA.

Duration00:10:00   
Temperature68 °C   

10m

Allow to slowly cool down from 68°C to 37°C for optimal hybridization.
To do this turn off the heat block and let it naturally fall down to 37°C

Preparation of beads

10m

Preparation of beads

Resuspend the streptavidin-coated magnetic beads (SMB) by carefully vortexing tube at medium speed.

For each sample transfer 90 μl  bead suspension  into a fresh tube.

For batch washing of beads for multiple samples, aliquot bead suspension for up to 6 (i.e. 540 μl) or 12 samples (i.e. 1080 μl) in a single tube.

Amount90 or X µL bead suspension  

Place tube on magnetic rack and wait for 1 min

Duration00:01:00   

Aspirate and discard all supernatant, while keeping the tube on the magnetic rack.

Add 80 μl per sample (i.e. 480 μl for 6 samples, 960 μl for 12 samples ) of Depletion Buffer (DB) and agitate the tube well to resuspend beads. 
Place on magnetic rack (1 min), aspirate and discard supernatant.

Amount80 µL Depletion Buffer (DB)  
Duration00:01:00   

Resuspend beads in 80 μl per sample (i.e. 480 μl 6 samples) of Depletion Buffer and agitate the tube well to resuspend beads.

Amount80 µL  Depletion Buffer (DB)  

Depletion

10m

Briefly centrifuge the tube containing ~20 μl hybridized riboPOOL and total RNA (from step 26.3) to spin down droplets.

Combine 80 µl of prepared beads (from step 27.5) with ~20 μl of the hybridized riboPOOL-RNA solution. Agitate the tube to resuspend well.

Amount80 µL  prepared beads  
Amount20 µL hybridized RNA  

Incubate at 37°C for 15 min, followed by a 50°C incubation for 5 min.

Temperature37 °C  15 min  
Temperature50 °C 5 min  

Briefly spin down droplets. Place on magnet for 2 min. Carefully transfer the supernatant to a new tube.

Duration00:02:00  Magnetic Rack  
Note
OBS: This new tube is where RNA will be precipitated, make sure to use the ones provided by the kit in order to get a clear visible pellet. 
If you are running the optional step below make sure to have this kind of tube for step 28.5

Place the new tube with the supernatant on the magnetic rack for 1 min to get rid of trace amounts of beads. (optional)

Duration00:01:00  Magnetic Rack  

Carefully transfer the supernatant to a new tube (optional).

Note
At this point, RNA can be stored at -20°C overnight or -80°C for up to a month according to the procol, however it's advisable to proceed with the RNA Purification step(29).

RNA Purification

10m

Clean up by EtOH precipitation
Can be done either over night at -20°C, or for 30 min at -80°C.


Temperature-20 °C   DurationOvernight   
Or
Temperature-80 °C   Duration00:30:00   

Add 10 µl of 3M sodium acetate (SA), 1 µl of Linear acrylamide (LA) and 333 µl of 100%
ethanol to the RNA solution from step 28.5

SampleSample  
Amount10 µL Sodium Acetate (SA)  
Amount1 µL  Linear acrylamide (LA)  
Amount333 µL EtOh (100%)  


Vortex the mixture well

Incubate tube at -80°C for 30 min OR -20°C overnight

AFTER INCUBATION:
Centrifuge at 11 000 x g (or max speed) for 30 min at 4°C

Centrifigation11000 rcf, 4°C, 00:30:00 ,  (Or at max speed)  

30m

Carefully remove and discard all supernatant, making sure not to disrupt pellet.

Add 200 μl of 70% ethanol to wash pellet.

 Amount200 µL EtOh (70%)  

Centrifuge at 11 000 x g for 5 min at 4°C

Centrifigation11000 rcf, 4°C, 00:05:00  

Carefully remove and discard all supernatant, making sure not to disrupt pellet.

Repeat ethanol wash (steps 29.6 to 29.8)

Dry pellet at room temperature for 5 min

Resuspend the pellet in 32 μl of nuclease-free water (H2O) or appropriate elution buffer for library preparation. If required, elution volume can be decreased down to 10 µl.

AmountX µL  (Or whatever your final end volume should be)  

Run a quality control on the depleted RNA.

Quality Control - BioAnalyzer

10m

Info: For QC control the BioAnalyzer was used in combination with the Agilent RNA 6000 Nano Kit. 
The protocol followed can be found here (Protocol).

For each sample to be analyzed 1 µl of dissolved RNA is needed.

Amount1 µL RNA sample  

Check whether there is fresh filtered Gel prepared or if new gel has to be prepared (Lasts 4 weeks)

Decontaminate the Electrodes:

Slowly fill one of the wells of an electrode cleaner with 350 µl RNaseZAP. 
 Open the lid and place the electrode cleaner in the Agilent 2100 Bioanalyzer. 
Close the lid and leave it closed for about 1 minute. 
Open the lid and remove the electrode cleaner. Label the electrode cleaner and keep it for future use. You can reuse the electrode cleaner for all 25 chips in the kit. 
Slowly fill one of the wells of another electrode cleaner with 350 µl RNase-free water.
Place the water-filled chip in the Agilent 2100 bioanalyzer, close the lid and leave for 10s.
Remove the chip and leave the lid open for 10s to dry the electrode. 

Run the gel according to manufacturers instructions
Use the assay "mRNA Nano Series II"

Protocol references

This RNA extraction is based on Quick-RNA Fecal/Soil Microbe Microprep Kit 
https://files.zymoresearch.com/protocols/_r2040_quick-rna_fecal-soil_microbe_microprep_kit.pdf ("Ensure you use the latest protocol version from the manufacturer's website.)

Quality control and concentration measurement procedure is described in user manual for Agilent RNA 6000 Nano Kit Guide. 
https://www.agilent.com/cs/library/usermanuals/Public/G2938-90034_RNA6000Nano_KG.pdf 

RNA depletion and RNA precipitation is based on the RiboPool kit (Pan-Prokayote) 
https://www.bioscience.co.uk/resources/ribopool-protocol.pdf

Public workspaceRNA extraction & depletion V.1

RNA extraction & depletion V.1