Jan 30, 2023

Public workspaceRNA-Stable Isotope Probing V.9

DOI
dx.doi.org/10.17504/protocols.io.kxygxm23kl8j/v9
RNA-Stable Isotope Probing
  • Roey Angel1,
  • Eva Petrova1,
  • Ana Lara1
  • 1Soil and Water Research Infrastructure
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: eva.petrova@bc.cas.cz
Roey Angel
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxm23kl8j/v9
Protocol Citation: Roey Angel, Eva Petrova, Ana Lara 2023. RNA-Stable Isotope Probing. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxm23kl8j/v9Version 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: January 30, 2023
Last Modified: January 30, 2023
Protocol Integer ID: 76053
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, extraction, 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.
Citation
Whiteley AS, Thomson B, Lueders T, Manefield M (2007). RNA stable-isotope probing. Nature protocols.
10.1038/nprot.2007.115
LINK

Citation
Angel R, Conrad R (2013). Elucidating the microbial resuscitation cascade in biological soil crusts following a simulated rain event. Environmental microbiology.
https://doi.org/10.1111/1462-2920.12140
LINK

Citation
Dumont MG, Pommerenke B, Casper P, Conrad R (2011). DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment. Environmental microbiology.
https://doi.org/10.1111/j.1462-2920.2010.02415.x
LINK

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.

Citation
Angel R (2019). Experimental Setup and Data Analysis Considerations for DNA- and RNA-SIP Experiments in the Omics Era. Methods in molecular biology (Clifton, N.J.).
https://doi.org/10.1007/978-1-4939-9721-3_1
LINK

Citation
Sieradzki ET, Koch BJ, Greenlon A, Sachdeva R, Malmstrom RR, Mau RL, Blazewicz SJ, Firestone MK, Hofmockel KS, Schwartz E, Hungate BA, Pett-Ridge J (2020). Measurement Error and Resolution in Quantitative Stable Isotope Probing: Implications for Experimental Design. mSystems.
https://doi.org/pii:e00151-20.10.1128/mSystems.00151-20
LINK

Citation
Youngblut ND, Barnett SE, Buckley DH (2018). HTSSIP: An R package for analysis of high throughput sequencing data from nucleic acid stable isotope probing (SIP) experiments. PloS one.
https://doi.org/10.1371/journal.pone.0189616
LINK

Citation
Hungate BA, Mau RL, Schwartz E, Caporaso JG, Dijkstra P, van Gestel N, Koch BJ, Liu CM, McHugh TA, Marks JC, Morrissey EM, Price LB (2015). Quantitative microbial ecology through stable isotope probing. Applied and environmental microbiology.
https://doi.org/10.1128/AEM.02280-15
LINK

Citation
Angel R, Panhölzl C, Gabriel R, Herbold C, Wanek W, Richter A, Eichorst SA, Woebken D (2018). Application of stable-isotope labelling techniques for the detection of active diazotrophs. Environmental microbiology.
https://doi.org/10.1111/1462-2920.13954
LINK

Materials
STEP MATERIALS
ReagentTrizma® hydrochloride / Tris-HClMerck MilliporeSigma (Sigma-Aldrich)Catalog #T5941
ReagentPotassium chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
ReagentEthylenediaminetetraacetic acid disodium salt dihydrate BioUltra 98.5-101.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644-100G
ReagentCesium Trifluoroacetate (CsTFA) illustra™Thermo Fisher ScientificCatalog #45-000-147
ReagentHi-Di FormamideThermo Fisher ScientificCatalog # 4311320
ReagentGlycoBlue™ coprecipitantThermo Fisher ScientificCatalog # AM9515
Reagent3M Na-Acetate pH 5.5Thermo Fisher ScientificCatalog # AM9740
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
ReagentRandom hexamersThermo ScientificCatalog #N8080127
ReagentBovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
ReagentdNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
ReagentSuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050
ReagentRNaseOUT™ 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
ReagentRNase AWAY™ Surface DecontaminantThermo Fisher ScientificCatalog #7002PK
ReagentEthylenediaminetetraacetic acid disodium salt dihydrate BioUltra 98.5-101.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644-100G
ReagentGlycoBlue™ coprecipitantThermo Fisher ScientificCatalog # AM9515
ReagentBovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
ReagentTrizma® hydrochloride / Tris-HClMerck MilliporeSigma (Sigma-Aldrich)Catalog #T5941
ReagentdNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191
ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
ReagentCesium Trifluoroacetate (CsTFA) illustra™Thermo Fisher ScientificCatalog #45-000-147
Reagent3M Na-Acetate pH 5.5Thermo Fisher ScientificCatalog # AM9740
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
ReagentRandom hexamersThermo ScientificCatalog #N8080127
ReagentPotassium chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
ReagentSuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050
ReagentHi-Di FormamideThermo Fisher ScientificCatalog # 4311320
ReagentCesium trifluoroacetateThermo Fisher ScientificCatalog #44633.18
ReagentCesium Trifluoroacetate Solution FUJIFILM Wako Pure Chemical CorporationCatalog #031-13681
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
  1. Prepare all buffers and solutions in advance (see Step 1).
  2. Wipe all surfaces and apparatus with an RNase eliminating solution (e.g. RNAse Away).
  3. Equilibrate CSTFA to room temperature (about 30-60 min).
  4. 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.

ReagentRNase 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 Temperature180 °C for Duration04:00:00


Gradient buffer (0.1 M Tris-HCl, 0.1 M KCl, 1 mM EDTA) Ph8.0 :
Amount15.76 g Tris-HCl
Amount7.455 g KCl
Amount0.37224 g EDTA

Dissolve the salts in RNase-free water and fill up toAmount1000 ml . Filter sterilise (0.1-0.2 μm). Autoclave.
ReagentTrizma® hydrochloride / Tris-HClMerck MilliporeSigma (Sigma-Aldrich)Catalog #T5941

ReagentPotassium chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9333

ReagentEthylenediaminetetraacetic acid disodium salt dihydrate BioUltra 98.5-101.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644-100G
Store atTemperatureRoom temperature
Molecular-grade ethanol solution (Concentration75 % (v/v) ):
Amount75 ml Absolute ethanol
Amount25 ml RNase-free water
Store at Temperature-20 °C



If preparing CsTFA solution from powder:
  1. PourAmount17.2 ml gradient buffer directly into aAmount50 g CsTFA vial.
  2. Mix by hand atTemperatureRoom temperature and then allow for the residues to fully dissolve during several hours or overnight.
  3. If particle impurities are visible, filter the solution into a fresh bottle using a 0.1 or 0.2 μm filter.
  4. Store atTemperature4 °C .

Note
1. CsTFA will not dissolve well in pure water, and the pH will be too low
2. CsTFA powder is highly hygroscopic. It is, therefore, recommended to dissolve the entire content of the bottle immediately.
3. It is recommended to dissolve the powder first, using only around Amount16.5 mL of gradient buffer and adjust it afterwards.
4. Confirm that the density is 2 g ml-1 using an analytical scale and adjust the solution if necessary.


ReagentCesium trifluoroacetateThermo Fisher ScientificCatalog #44633.18

Optional
Gradient preparation
1h
Calibrate the refractometer with RNAse-free water atTemperature20 °C .
Amount30 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
https://www.reichertai.com/products/handheld-digital/ar200-automatic-digital-refractometer/
LINK

For every two gradients, mix the following in a 50 ml tube (assuming 6 ml Ultracrimp, PA centrifugation tubes):
Amount9.696 ml CsFTA
Amount2.166 ml Gradient Buffer
TemperatureRoom temperature
Note
Adjust the volumes if using different-sized ultracentrifugation tubes.

ReagentCesium Trifluoroacetate Solution FUJIFILM Wako Pure Chemical CorporationCatalog #031-13681

Equipment
Thermo Scientific TUBE PA ULTRACRIMP 6ML PK/50
NAME
Ultracentrifugation tubes
TYPE
Thermo Fisher Scientific
BRAND
03945
SKU
https://www.fishersci.com/shop/products/tube-pa-ultracrimp-6ml-pk-50/03945
LINK


Toxic
Mix by inverting several times, pipetteAmount30 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.

AddConcentration3.56 % (v/v) HiDi (Amount422 µL if the volume was not corrected).

ReagentHi-Di FormamideThermo Fisher ScientificCatalog # 4311320

Toxic
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 ca.Amount5.8 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.
Amount4 µL RNA (1-8 µl)
Concentration150 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
https://www.fishersci.com/shop/products/tool-ultracrimp-ea/03920
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
Centrifigation130000 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.

Centrifigation
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
https://online-shop.eppendorf.com/OC-en/Laboratory-Consumables-44512/Tubes-44515/DNA-LoBind-Tubes-PF-56252.html
LINK

Fill a 20 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
https://www.syringepump.com/NE-300.php
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
https://www.coleparmer.com/i/masterflex-l-s-precision-pump-tubing-peroxide-cured-silicone-l-s-14-25-ft/9640014
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
https://www.coleparmer.com/i/masterflex-fitting-polycarbonate-straight-female-luer-to-low-profile-semi-rigid-barb-hose-adapter-1-8-id-25-pk/4550116
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
coleparmer.com/i/masterflex-fitting-polypropylene-straight-male-luer-lock-to-hose-barb-adapter-1-16-id-25-pk/3080016
LINK

Equipment
Disposable needles Sterican® long bevel facet, 30 mm, 0.60 mm, Blue
NAME
Disposable needles
TYPE
Sterican
BRAND
X129.1
SKU
https://www.carlroth.com/com/en/starter-kit-dna-synthesis-for-approx-100x-30mers/disposable-needles-sterican-long-bevel-facet/p/x129.1
LINK

Set the Rate to 1 ml min-1 and collect fractions inDuration00:00:30 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 20 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.

Critical
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
https://www.carlroth.com/com/en/starter-kit-dna-synthesis-for-approx-100x-30mers/disposable-needles-sterican-long-bevel-facet/p/c718.1
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 Duration00:00:30 (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.
Amount30 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 range between 1.75 and 1.84 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.


Imaging
RNA precipitation
2h
To each tube add Amount2 µL of GlycoBlue, Concentration10 % (v/v) Na-Acetate (Concentration3 Molarity (M) ), and Concentration250 % (v/v) of absolute ethanol. Assuming Amount500 µL fractions were collected and Amount30 µL were spent for determining the density, add Amount47 µL Na-acetate and Amount1175 µL ethanol (absolute) .
ReagentGlycoBlue™ coprecipitantThermo Fisher ScientificCatalog # AM9515

Reagent3M Na-Acetate pH 5.5Thermo Fisher ScientificCatalog # AM9740

Note
GlycoBlue is particularly advantageous here because otherwise, the pellet is completely invisible.


Pipetting
Incubate at Temperature-80 °C for Duration00:30:00 .

Incubation
Centrifuge at Centrifigation14000 rpm, 4°C, 00:30:00 .

Centrifigation
Decant the supernatant, wash once with Amount1 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.



Wash
Centrifuge at Centrifigation14000 rpm, 4°C, 00:10:00 .

Centrifigation
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.
Duration00:05:00 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 Amount10 µL RNase-free water or the RNA Storage solution.
ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000

cDNA synthesis
2h
For each fraction, prepare the following mixture in a PCR tube:
  1. Amount10 µL template RNA
  2. Amount3 µL random hexamers (Concentration50 micromolar (µM) diluted 20x in RNase-free water: Concentration2.5 micromolar (µM) )

ReagentRandom hexamersThermo ScientificCatalog #N8080127

Incubate the mixture at Temperature65 °C for Duration00:05:00 in a thermocycler and chill at Temperature4 °C for at least Duration00:01:00 .

Prepare the following mixture (times the number of fractions) and add Amount7 µL into each tube:

  1. Amount4 µL 5x Reaction buffer
  2. Amount1 µL 10 mM dNTP mix
  3. Amount1 µL 0.1 M DTT (optional)
  4. Amount0.2 µL RNase OUT (40 U/µl; optional)
  5. Amount0.2 µL BSA (20 µg/µl)
  6. Amount0.1 µL SuperScript IV RT (200 U/µl)
  7. Amount0.5 µL RNase-free water
ReagentSuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050

ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019

ReagentBovine Serum Albumin (BSA)Thermo Fisher ScientificCatalog #B14

ReagentdNTP Mix (10 mM each)Thermo Fisher ScientificCatalog #R0191

ReagentUSB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML

Incubate the mixture in a thermocycler for Duration00:10:00 at Temperature23 °C followed by Duration01:00:00 at Temperature50 °C and then Duration00:10:00 at Temperature80 °C . Chill at Temperature4 °C .
Dilute Amount1 µL cDNA in Amount14 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

Protocol
qPCR: Bacterial SSU rRNA 338F-516P-805R
NAME

qPCR: Bacterial SSU rRNA 338F-516P-805R

CREATED BY
Roey Angel

Pipetting

NameTypeSequenceTarget region1
BAC338FForwardACT CCT ACG GGA GGC AG338-354
BAC516P2ProbeTGC CAG CAG CCG CGG TAA TA516-536
BAC805RReverseGAC 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
ReagentFinal concentration1 tube (20 μl)plate (20 μl x 100)
PCR H2O 4.6460
iQTM Supermix1x101000
MgCl2 (25 mM)4.0 mM0.8180
BSA (20 μg μl-1)0.2 μg μl-10.220
338F (10 μM)0.5 μM1.0100
805R (10 μM)0.5 μM1.0100
516P (10 μM)0.2 μM0.440
Template 22 x 100
1 Buffer contains  MgCl2 at final conc. of 3.0 mM

  1. Temperature95 °C for Duration00:05:00
  2. x 40 {
2.1 Temperature95 °C for Duration00:00:30
2.2 Temperature62 °C forDuration00:00:30 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
An example of successful labelling with 13C, seen via the presence of a peak in the copy numbers around 1.82 g ml-1
Download Plot_SIP_example.RMDPlot_SIP_example.RMD Download Frac_density_example.csvFrac_density_example.csv Download qPCR_SIP_example.csvqPCR_SIP_example.csv
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
Youngblut ND, Barnett SE, Buckley DH (Invalid date). HTSSIP: An R package for analysis of high throughput sequencing data from nucleic acid stable isotope probing (SIP) experiments. PloS one.
https://doi.org/10.1371/journal.pone.0189616
LINK

Citation
Angel R (Invalid date). Experimental Setup and Data Analysis Considerations for DNA- and RNA-SIP Experiments in the Omics Era. Methods in molecular biology (Clifton, N.J.).
https://doi.org/10.1007/978-1-4939-9721-3_1
LINK






Computational step
Citations
Whiteley AS, Thomson B, Lueders T, Manefield M. RNA stable-isotope probing
10.1038/nprot.2007.115
Angel R, Conrad R. Elucidating the microbial resuscitation cascade in biological soil crusts following a simulated rain event.
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