Nov 23, 2020
Version 3
RNA-Stable Isotope Probing V.3
- Roey Angel1,
- Eva Petrova1,
- Ana Lara1
- 1Soil and Water Research Infrastructure
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: [email protected]
Protocol Citation: Roey Angel, Eva Petrova, Ana Lara 2020. RNA-Stable Isotope Probing. protocols.io https://dx.doi.org/10.17504/protocols.io.bpwympfwVersion created by Roey Angel
Manuscript citation:
Angel, R., and Conrad, R. (2013). Elucidating the microbial resuscitation cascade in biological soil crusts following a simulated rain event. Environ Microbiol 15, 2799–2815. doi:10.1111/1462-2920.12140.
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 20, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44728
Keywords: Stable isotope probing, SIP, RNA, Ultracentrifugation, Density gradient, protocol total nucleic acids extraction from soil, protocol total nucleic acids extraction, purification of rna, extracting rna, stable isotope probing, separation of the rna, stable isotope probing experiment, labelled rna, nitrogen in the rna, stable isotope, rna from unlabelled rna, rna, unlabelled rna, crude na extract protocol, total rna, purification, environmental sample, labelled substrate, nitrogen, downstream quantification, sip experiment,
Abstract
The following protocol describes how to perform an RNA-Stable Isotope Probing experiment. The scope of this protocol only covers the parts involving separating labelled RNA from unlabelled RNA using ultracentrifugation in a caesium trifluoroacetate density gradient and downstream quantification to evaluate whether the labelling and separation of the RNA were successful. Total RNA should be extracted from an environmental sample or an enrichment culture that was incubated with an isotopically-labelled substrate. Labelling can be of the carbon, oxygen or nitrogen in the RNA (or any combination of the 3). For environmental samples, we recommend extracting RNA using our protocol Total Nucleic Acids Extraction from Soil and purifying it using the Purification of RNA from Crude NA Extract protocol. This protocol is based on the following papers: Whiteley et al. (2007); Dumont et al. (2011); Angel and Conrad (2013). For a comprehensive discussion on how to design a SIP experiment and how to analyse the resulting data, we recommend referring to the recent book on the subject: Stable Isotope Probing: Methods and Protocols, especially chapters: 1-3 and 9-18.
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Guidelines
- Design of SIP experiments. SIP experiments are usually relatively complex, laborious, and time-consuming, and can, therefore, fail because of various reasons and at different stages. Therefore, the design of a SIP experiment should be carefully considered in advance and cover all aspects and phases, including preliminary knowledge of the environment and the targeted process, the nature and duration of the incubation, how many and what types of controls to include, how many fractions to collect and how deep to sequence. These considerations extend beyond the scope of this protocol. Comprehensive discussions and tips on how to best design a SIP experiment can be found at Angel (2019) and Sieradzki et al. (2020).
- RNA handling. Since RNA is very sensitive to both chemical and enzymatic degradation, some precautionary measures should be taken. The RNA molecules are protected from degradation while in the CsTFA gradient but are sensitive to degradation during the precipitation and washing steps and downstream applications. For more info see Total Nucleic Acids Extraction from Soil.
- Reducing the volume required for the refractometer. The typical handheld-refractometer such as the Reichert AR200 has a large lens size requiring 50-100 µl of liquid to cover its surface adequately. To minimise the volume of wasted sample, it is possible to cover the lens with a piece of strong dark adhesive tape, to which a hole was made using a perforator.
- Timing. The timings for each step listed SIP protocol assume that only two gradients are being processed simultaneously. We recommend processing more than 4-8 gradients at a time, but not more.
- Data analysis. Several statistical frameworks have been developed in recent years to analyse SIP datasets such as qSIP (Hungate et al., 2015), HR-SIP (Youngblut et al., 2018) and HR-RNA-SIP (Angel et al., 2018).
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Materials
STEP MATERIALS
Trizma® hydrochloride / Tris-HClMerck MilliporeSigma (Sigma-Aldrich)Catalog #T5941
Potassium chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Ethylenediaminetetraacetic acid disodium salt dihydrate BioUltra 98.5-101.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644-100G
Cesium Trifluoroacetate (CsTFA) illustra™Thermo Fisher ScientificCatalog #45-000-147
Hi-Di FormamideThermo Fisher ScientificCatalog # 4311320
GlycoBlue™ coprecipitantThermo Fisher ScientificCatalog # AM9515
3M Na-Acetate pH 5.5Thermo Fisher ScientificCatalog # AM9740
THE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
Random hexamersThermo ScientificCatalog #N8080127
Bovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
dNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
USB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
SuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Apparatus
1) For gradient preparation
- Working bench in a climatised room at 20 °C
- Icebox
- 50 ml tube (for up to 8 gradients)
- Ultracentrifuge (capable of achieving 177,000 g) and a vertical rotor (e.g. Sorvall WX Ultra 100 Ultracentrifuge, TV-1665 rotor). A fixed-angle
- Ultracentrifugation tubes (e.g. Ultracrimp, PA centrifugation tubes 6 ml)
- Ultracentrifugation tube caps
- Refractometer
- Purified RNA samples (DNA-free) with a concentration >20 ng µl-1
- Micropipettes and tips
- Lab-scale
2) For fractionation
- Working bench in a climatised room at 20°C
- Refractometer
- Low-binding tubes (one per fraction; 1.5 ml)
- Test tube utility clamp mounted on a stand
- Automatic syringe pump (e.g. NewEra's NE-300 Syringe Pump)
- 20 ml syringe
- Precision pump peroxide-cured silicone tube (or similar), 1/16'', about 0.5-1 m long
- Luer fittings (1/16''), male and female, to fit the tube on a syringe on one end and a disposable needle on the other end
- Disposable syringe needles: 23G and 26G
- Stopwatch
- Micropipettes and tips
Protocol materials
RNase AWAY™ Surface DecontaminantThermo Fisher ScientificCatalog #7002PK
THE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
SuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050
GlycoBlue™ coprecipitantThermo Fisher ScientificCatalog # AM9515
USB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
Potassium chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Ethylenediaminetetraacetic acid disodium salt dihydrate BioUltra 98.5-101.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644-100G
Cesium Trifluoroacetate (CsTFA) illustra™Thermo Fisher ScientificCatalog #45-000-147
Hi-Di FormamideThermo Fisher ScientificCatalog # 4311320
Random hexamersThermo ScientificCatalog #N8080127
Bovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
Trizma® hydrochloride / Tris-HClMerck MilliporeSigma (Sigma-Aldrich)Catalog #T5941
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
3M Na-Acetate pH 5.5Thermo Fisher ScientificCatalog # AM9740
dNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
Troubleshooting
Safety warnings
Safety information
CsTFA is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).
Causes respiratory tract, eye and skin irritation. May be harmful if swallowed.
Do not ingest. Avoid breathing vapour or mist. Use only with adequate ventilation. Avoid contact with eyes, skin and clothing. Keep container tightly closed. Wash thoroughly after handling.
HiDi-formamide may damage fertility or the unborn child if swallowed. Suspected of causing cancer if swallowed. May cause damage to organs through prolonged or repeated exposure.
Do not breathe fumes or spray. Wear protective gloves/protective clothing/eye protection/face protection.
- Storage and waste
- Store below eye level to prevent injuries in case of a spill.
- Dispose of CsTFA and HiDi-formamide in a sealed container as hazardous waste.
Before start
- Wipe all surfaces and apparatus with an RNase eliminating solution (e.g. RNAse Away).
- Equilibrate CSTFA to room temperature (about 30-60 min).
- Prepare one 50 ml tube (for up to 8 gradients; depending on the size of the centrifugation tubes) and one ultracentrifugation tube for each gradient.
RNase AWAY™ Surface DecontaminantThermo Fisher ScientificCatalog #7002PK
Solutions for SIP
1h
Prepare the following solutions:
Note
All glassware and plasticware must be clean and free of RNA and RNAse. Glassware can be baked at 180 °C for 04:00:00
Gradient buffer (0.1 M Tris-HCl, 0.1 M KCl, 1 mM EDTA) 8.0 :
15.76 g Tris-HCl
7.455 g KCl
0.37224 g EDTA
Dissolve the salts in RNase-free water and fill up to 1000 ml . Filter sterilise (0.1-0.2 μm). Autoclave.
Trizma® hydrochloride / Tris-HClMerck MilliporeSigma (Sigma-Aldrich)Catalog #T5941
Potassium chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Ethylenediaminetetraacetic acid disodium salt dihydrate BioUltra 98.5-101.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644-100G
Store at Room temperature
Molecular-grade ethanol solution (75 % (v/v) ):
75 ml Absolute ethanol
25 ml RNase-free water
Store at -20 °C
Gradient preparation
1h
Calibrate the refractometer with RNAse-free water at 20 °C .
30 µL RNAse-free water
Following calibration, the device should read 1.3330 ± 0.0002 nD-TC
Equipment
AR200 Automatic Digital Refractometer
NAME
Digital Refractometer
TYPE
Reichert
BRAND
13950000
SKU
LINK
For every two gradients, mix the following in a 50 ml tube (assuming 6 ml Ultracrimp, PA centrifugation tubes):
9.696 ml CsFTA
2.166 ml Gradient Buffer
Room temperature
Note
Adjust the volumes if using different-sized ultracentrifugation tubes.
Cesium Trifluoroacetate (CsTFA) illustra™Thermo Fisher ScientificCatalog #45-000-147
Equipment
Thermo Scientific TUBE PA ULTRACRIMP 6ML PK/50
NAME
Ultracentrifugation tubes
TYPE
Thermo Fisher Scientific
BRAND
03945
SKU
LINK
Mix by inverting several times, pipette 30 µL and measure the density in a refractometer. Make sure the density is: 1.3702 ± 0.0002 nD-TC. Otherwise, add either CsTFA or GB to correct.
Add 3.56 % (v/v) HiDi (422 µL if the volume was not corrected).
Hi-Di FormamideThermo Fisher ScientificCatalog # 4311320
Measure the density. Make sure the density is: 1.3725 ± 0.0002 nD-TC.
Note
Due to potential variability between batches, it is recommended to add a slightly lower volume of HiDi at first to avoid exceeding the recommended refractive index.
Transfer approx. 5.8 ml of the mixture to each centrifugation tube using a micropipette. Make sure the volume reaches only the bottom of the neck.
Add the RNA sample. For downstream PCR purposes, ca. 200-350 ng is more than enough. Preferably, use a highly concentrated RNA solution to avoid diluting the gradient.
4 µL RNA (1-8 µl)
150 ng/µl RNA (75-600 ng)
Note
The amount of RNA should not exceed 100 ng per 1 ml of gradient mixture.
Weigh each tube together with the caps and make sure every opposite pair of tubes weighs no more than 0.1 g apart from each other. Otherwise, adjust the weight by adding gradient mix solution.
Close the caps (using an appropriate crimper or by hand, depending on the type of tubes).
Equipment
Thermo Scientific TOOL ULTRACRIMP EA
NAME
Tube crimper
TYPE
Thermo Fisher Scientific
BRAND
03920
SKU
LINK
Place the tubes in the rotor, screw only the caps for the positions that contain tubes using a torque wrench up to about 120 in.-lb.
Ultracentrifugation
2d 17h
Centrifuge
130000 x g, 20°C, 65:00:00 , (37,900 rpm for the TV-1665 rotor)
Maximum acceleration and deceleration.
Note
Because the density gradient will stabilise over time, centrifuging for a longer time period will make no difference but can be used for timing reasons. However, after the centrifugation has stopped the gradient will slowly diffuse back to its original state. Therefore, the gradients are best fractionated immediately.
Fractionation
1h
Prepare a rack filled with 2.0 ml low-binding collection tubes (one per fraction).
Equipment
DNA LoBind Tubes
NAME
Microcentrifuge tubes
TYPE
Eppendorf
BRAND
0030108051
SKU
LINK
Fill a 30 ml syringe with RNase-free water. Remove any air bubbles.
Attach a female Luer fitting to one end of a precision pump tube (about 0.5 m long) and a male Luer fitting to the other end. Attach the syringe to the precision pump tube on the female Luer fitting side. Attach a sterile 23G needle to the other end of the tube on the male Luer fitting side. Lightly press the syringe piston to get water into the tube and mount the syringe on an automatic syringe pump.
Equipment
NE-300 Just Infusion™ Syringe Pump
NAME
Automatic syringe pump
TYPE
New Era Pump Systems, Inc.
BRAND
NE-300
SKU
LINK
Equipment
Masterflex L/S® Precision Pump Tubing, Peroxide-Cured Silicone, L/S 14; 25 ft
NAME
Silicone tube
TYPE
Masterflex
BRAND
96400-14
SKU
LINK
Equipment
Masterflex Fitting, Polycarbonate, Straight, Female Luer to Low-Profile Semi-Rigid Barb Hose Adapter, 1/16" ID; 25/PK
NAME
Luer fitting
TYPE
Masterflex
BRAND
45501-16
SKU
LINK
Equipment
Masterflex Fitting, Polypropylene, Straight, Male Luer Lock to Hose Barb Adapter, 1/16" ID; 25/PK
NAME
Luer fitting
TYPE
Masterflex
BRAND
30800-16
SKU
LINK
Equipment
Disposable needles Sterican® long bevel facet, 30 mm, 0.60 mm, Blue
NAME
Disposable needles
TYPE
Sterican
BRAND
X129.1
SKU
LINK
Set the Rate to 1 ml min-1 and collect fractions in 00:00:30 steps. If using a 6 ml tube, this will yield 12 fractions. Volume should be set to "off" and diameter to "22 mm".
Note
For collecting more or fewer fractions, adjust the speed or collection rate.
Note
Using a different syringe (other than 30 ml) will require adjusting the inner diameter setting on the pump
30s
Switch the pump on to test the system and also to get rid of air trapped inside the needle and any air bubbles in the tube. Switch the pump back off.
Stop the ultracentrifuge. Remove the rotor and open the screw-caps. Take the first tube out of the rotor and carefully mount it on a stand with a clamp holder just above the collection tubes.
Note
Make sure the tube stays upright during handling.
Pierce the ultracentrifugation tube, just below the neck, using the needle attached to to the precision pump tube.
Note
Be careful not to pierce through the other end of the tube!
If the other end of the tube was accidentally pierced, a small amount of petroleum gel can be used to seal the hole.
Take a new, sterile 26G needle, carefully puncture a hole at the bottom of the ultracentrifugation tube and remove the needle. The tube should not leak at this stage.
Equipment
Disposable needles Sterican® long bevel facet, 25 mm, 0.45 mm, Brown
NAME
Disposable needles
TYPE
Sterican
BRAND
c718.1
SKU
LINK
Open all the collection tubes in the rack and make sure the first tube is positioned just below the bottom hole of the ultracentrifugation tube.
Your set-up should look like this:
The SIP fraction collection set-up ready to start
Start the pump, as soon as the first drop falls off the ultracentrifugation tube start the stopwatch
After 00:00:30 (or your chosen time interval), shift the rack so that the drops will fall into the second collection tube. Continue in a similar fashion until all tubes have been filled. Close the tubes to avoid contamination and label them.
30s
Measure the density of each fraction using the refractometer. Start from the last (the lightest) fraction.
30 µL of each fraction
The density of the fractions should increase at a linear rate as you progress from the lighter to the heavier fraction.
The conversion between refractive index (n) to density (ρ) is (empirically):
And can be easily determined in the lab by weighing a known volume of several fractions and establishing a calibration curve.
The gradient should be in the range of 1.77-1.80 g ml-1, assuming a vertical rotor was used (a fixed-angle rotor will yield a steeper gradient, meaning a wider range of densities).
Note
Typically the first and last fractions are discarded because they contain little to no nucleic acids.
RNA precipitation
2h
To each tube add 2 µL of GlycoBlue, 10 % (v/v) Na-Acetate (3 Molarity (M) ), and 250 % (v/v) of absolute ethanol. Assuming 500 µL fractions were collected and 30 µL were spent for determining the density, add 47 µL Na-acetate and 1175 µL ethanol (absolute) .
GlycoBlue™ coprecipitantThermo Fisher ScientificCatalog # AM9515
3M Na-Acetate pH 5.5Thermo Fisher ScientificCatalog # AM9740
Note
GlycoBlue is particularly advantageous here because otherwise, the pellet is completely invisible.
Incubate at -80 °C for 00:30:00 .
Centrifuge at 14000 rpm, 4°C, 00:30:00 .
Decant the supernatant, wash once with 1 ml 75% ethanol, ice-cold , invert the tube several times.
Note
The pellet should be stable at this point and not detach from the tube's wall.
Centrifuge at 14000 rpm, 4°C, 00:10:00 .
Remove as much as possible from the supernatant first using a 1 ml tip, spin down the remaining drops in the tube, and remove them with a 100 μl tip.
Note
The pellet is unstable at this point. Be careful not to pipette the pellet with the liquid!
Leave the tubes open at room temperature for around 5 min (preferably under a flame or in a laminar-flow hood) in order to evaporate the remaining ethanol. Alternatively, the pellets can be dried under a filtered stream of air.
00:05:00 maximum time for drying
Note
The pellets might not be completely dry at this point, but the remaining liquid should be pure water.
Resuspend the pellets in 10 µL RNase-free water or the RNA Storage solution.
THE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
cDNA synthesis
2h
For each fraction, prepare the following mixture in a PCR tube:
- 10 µL template RNA
- 3 µL random hexamers (50 micromolar (µM) diluted 20x in RNase-free water: 2.5 micromolar (µM) )
Random hexamersThermo ScientificCatalog #N8080127
Incubate the mixture at 65 °C for 00:05:00 in a thermocycler and chill at 4 °C for at least 00:01:00 .
Prepare the following mixture (times the number of fractions) and add 7 µL into each tube:
- 4 µL 5x Reaction buffer
- 1 µL 10 mM dNTP mix
- 1 µL 0.1 M DTT (optional)
- 0.2 µL RNase OUT (40 U/µl; optional)
- 0.2 µL BSA (20 µg/µl)
- 0.1 µL SuperScript IV RT (200 U/µl)
- 0.5 µL RNase-free water
SuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Bovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
dNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
USB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
Incubate the mixture in a thermocycler for 00:10:00 at 23 °C followed by 01:00:00 at 50 °C and then 00:10:00 at 80 °C . Chill at 4 °C .
Dilute 1 µL cDNA in 14 µL RNase-free water for use as qPCR template. No dilution is required for use as a PCR template.
Note
This dilution step here is required to not exceed the range of detection of the qPCR assay. Higher or lower dilutions might be required depending on the amount of RNA that was loaded on the gradient and the recovery efficiency.
Evaluate the level of enrichment
2h 30m
Evaluate the level of isotopic enrichment using a qPCR assay. We recommend
| Name | Type | Sequence | Target region1 | |
| BAC338F | Forward | ACT CCT ACG GGA GGC AG | 338-354 | |
| BAC516P2 | Probe | TGC CAG CAG CCG CGG TAA TA | 516-536 | |
| BAC805R | Reverse | GAC TAC CAG GGT ATC TAA TC | 785-805 |
1. Relative to E. coli SSU rRNA gene
2. The probe must be dual-labelled either with 5’-6-FAM, 3’-BHQ1 or any other valid combination
| Reagent | Final concentration | 1 tube (20 μl) | plate (20 μl x 100) | |
| PCR H2O | 4.6 | 460 | ||
| iQTM Supermix | 1x | 10 | 1000 | |
| MgCl2 (25 mM) | 4.0 mM | 0.81 | 80 | |
| BSA (20 μg μl-1) | 0.2 μg μl-1 | 0.2 | 20 | |
| 338F (10 μM) | 0.5 μM | 1.0 | 100 | |
| 805R (10 μM) | 0.5 μM | 1.0 | 100 | |
| 516P (10 μM) | 0.2 μM | 0.4 | 40 | |
| Template | 2 | 2 x 100 |
1 Buffer contains MgCl2 at final conc. of 3.0 mM
- 95 °C for 00:05:00
- x 40 {
2.1 95 °C for 00:00:30
2.2 62 °C for00:00:30 take snapshot
}
Plot the cDNA copy numbers against the density of each fraction. It is common to normalise the qPCR results to the highest copy number in the gradient or to the total copy numbers of all the fractions in the gradient.
Expected result
Expect a peak of unlabelled RNA at around 1.78 g ml-1 and a peak of labelled RNA at around 1.82 g ml-1
Note
If the amount of labelled RNA is too small it might not be visible through qPCR. However, it might still be detectable through qSIP or HT-SIP analysis (see e.g. Youngblut et al., 2018, Angel, 2019)
Citation
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Citation
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Citations
Angel R, Conrad R. Elucidating the microbial resuscitation cascade in biological soil crusts following a simulated rain event.
https://doi.org/10.1111/1462-2920.12140Dumont MG, Pommerenke B, Casper P, Conrad R. DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment.
https://doi.org/10.1111/j.1462-2920.2010.02415.xWhiteley AS, Thomson B, Lueders T, Manefield M. RNA stable-isotope probing
10.1038/nprot.2007.115Angel R, Panhölzl C, Gabriel R, Herbold C, Wanek W, Richter A, Eichorst SA, Woebken D. Application of stable-isotope labelling techniques for the detection of active diazotrophs
https://doi.org/10.1111/1462-2920.13954Angel R. Experimental Setup and Data Analysis Considerations for DNA- and RNA-SIP Experiments in the Omics Era
https://doi.org/10.1007/978-1-4939-9721-3_1Sieradzki ET, Koch BJ, Greenlon A, Sachdeva R, Malmstrom RR, Mau RL, Blazewicz SJ, Firestone MK, Hofmockel KS, Schwartz E, Hungate BA, Pett-Ridge J. Measurement Error and Resolution in Quantitative Stable Isotope Probing: Implications for Experimental Design
https://doi.org/pii:e00151-20.10.1128/mSystems.00151-20Youngblut ND, Barnett SE, Buckley DH. HTSSIP: An R package for analysis of high throughput sequencing data from nucleic acid stable isotope probing (SIP) experiments
https://doi.org/10.1371/journal.pone.0189616Hungate BA, Mau RL, Schwartz E, Caporaso JG, Dijkstra P, van Gestel N, Koch BJ, Liu CM, McHugh TA, Marks JC, Morrissey EM, Price LB. Quantitative microbial ecology through stable isotope probing
https://doi.org/10.1128/AEM.02280-15