TITLE: RNA-Stable Isotope Probing
AUTHORS: Roey Angel, Eva Petrova, Ana Lara

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 </span><a href="https://www.protocols.io/view/total-nucleic-acids-extraction-from-soil-bi46kgze" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Total Nucleic Acids Extraction from Soil</span></a><span> and purifying it using the </span><a href="https://www.protocols.io/view/purification-of-rna-from-a-dna-rna-extract-bftbjnin" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Purification of RNA from Crude NA Extract</span></a><span> protocol. This protocol is based on the following papers: </span><a href="http://dx.doi.org/10.1038/nprot.2007.115" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Whiteley et al. (2007)</span></a><span>; </span><a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2010.02415.x/abstract" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Dumont et al. (2011)</span></a><span>; </span><a href="http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12140/abstract" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Angel and Conrad (2013).</span></a><span> 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: </span><a href="http://link.springer.com/10.1007/978-1-4939-9721-3" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Stable Isotope Probing: Methods and Protocols</span></a><span>, especially chapters: 1-3 and 9-18.</span></div><br/><br/><br/></div>

[STEPS]
?. [Solutions for SIP]
Prepare the following solutions:
All glassware and plasticware must be clean and free of RNA and RNAse. Glassware can be baked at   for
?. [Solutions for SIP]
Gradient buffer (0.1 M Tris-HCl, 0.1 M KCl, 1 mM EDTA)  :Dissolve the salts in RNase-free water and fill up to. Filter sterilise (0.1-0.2 μm). Autoclave.Store at
[Tris-HCl]
[KCl]
[EDTA]
1000
0 Room temperature
?. [Solutions for SIP]
Molecular-grade ethanol solution ():Store at
[Absolute ethanol]
[RNase-free water]
-20 °C
?. [Solutions for SIP]
If preparing CsTFA solution from powder:Pour directly into a vial.Mix by hand atand then allow for the residues to fully dissolve during several hours or overnight.Store at.
[gradient buffer]
[CsTFA]
0 Room temperature
4 °C
1. CsTFA powder will not dissolve well in pure water and the pH will be too low2. CsTFA powder is highly hygroscopic. It is, therefore, recommended to immediately dissolve the entire content of the bottle.
?. [Gradient preparation]
Calibrate the refractometer with RNAse-free water at .Following calibration, the device should read 1.3330 ± 0.0002 nD-TC
20 °C
[RNAse-free water]
?. [Gradient preparation]
For every two gradients, mix the following in a 50 ml tube (assuming 6 ml Ultracrimp, PA centrifugation tubes):
[CsFTA]
[Gradient Buffer]
65 Room temperature
Adjust the volumes if using different-sized ultracentrifugation tubes.
?. [Gradient preparation]
Mix by inverting several times, pipette 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.
30 µl
?. [Gradient preparation]
Add  HiDi ( if the volume was not corrected).
422 µl
?. [Gradient preparation]
Measure the density.  Make sure the density is: 1.3725 ± 0.0002 nD-TC.
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.
?. [Gradient preparation]
Transfer ca. of the mixture to each centrifugation tube using a micropipette. Make sure the volume reaches only the bottom of the neck.
5.8
?. [Gradient preparation]
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.
[RNA (1-8 µl)]
[RNA (75-600 ng)]
The amount of RNA should not exceed 100 ng per 1 ml of gradient mixture.
?. [Gradient preparation]
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.
?. [Gradient preparation]
Close the caps (using an appropriate crimper or by hand, depending on the type of tubes).
?. [Gradient preparation]
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]
Centrifuge Maximum acceleration and deceleration.
Centrifuge: 130000 34, 3900 min, 20 10
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]
Prepare a rack filled with 2.0 ml low-binding collection tubes (one per fraction).
?. [Fractionation]
Fill a 20 ml syringe with RNase-free water. Remove any air bubbles.
?. [Fractionation]
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.
?. [Fractionation]
Set the Rate to 1 ml min-1 and collect fractions in steps. If using a 6 ml tube, this will yield 12 fractions. Volume should be set to "off" and diameter to "22 mm".
For collecting more or fewer fractions, adjust the speed or collection rate.
Using a different syringe (other than 20 ml) will require adjusting the inner diameter setting on the pump
?. [Fractionation]
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.
?. [Fractionation]
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.
Make sure the tube stays upright during handling.
?. [Fractionation]
Pierce the ultracentrifugation tube, just below the neck, using the needle attached to to the precision pump tube.
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.
?. [Fractionation]
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.
?. [Fractionation]
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:
?. [Fractionation]
Start the pump, as soon as the first drop falls off the ultracentrifugation tube start the stopwatch
?. [Fractionation]
After  (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.
?. [Fractionation]
Measure the density of each fraction using the refractometer. Start from the last (the lightest) 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):ρ = 31.495n-41.439And 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).
[of each fraction]
Typically the first and last fractions are discarded because they contain little to no nucleic acids.
?. [RNA precipitation]
To each tube add  of GlycoBlue,  Na-Acetate ( ), and  of absolute ethanol. Assuming fractions were collected and   were spent for determining the density, add  Na-acetate and .
2 µl
500 µl
30 µl
47 µl
[ethanol (absolute)]
GlycoBlue is particularly advantageous here because otherwise, the pellet is completely invisible.
?. [RNA precipitation]
Incubate at   for .
-80 °C
?. [RNA precipitation]
Centrifuge at .
Centrifuge: 14000 33, 30 min, 4 10
?. [RNA precipitation]
Decant the supernatant, wash once with , invert the tube several times.
[75% ethanol, ice-cold]
The pellet should be stable at this point and not detach from the tube's wall.
?. [RNA precipitation]
Centrifuge at .
Centrifuge: 14000 33, 10 min, 4 10
?. [RNA precipitation]
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.
The pellet is unstable at this point. Be careful not to pipette the pellet with the liquid!
?. [RNA precipitation]
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.
[maximum time for drying]
The pellets might not be completely dry at this point, but the remaining liquid should be pure water.
?. [RNA precipitation]
Resuspend the pellets in  RNase-free water or the RNA Storage solution.
10 µl
?. [cDNA synthesis]
For each fraction, prepare the following mixture in a PCR tube:( diluted 20x in RNase-free water: )
[template RNA]
[random hexamers]
?. [cDNA synthesis]
Incubate the mixture at  for  in a thermocycler and chill at   for at least .
65 °C
4 °C
?. [cDNA synthesis]
Prepare the following mixture (times the number of fractions) and add  into each tube:
7 µl
[5x Reaction buffer]
[10 mM dNTP mix]
[0.1 M DTT (optional)]
[RNase OUT (40 U/µl; optional)]
[BSA (20 µg/µl)]
[SuperScript IV RT (200 U/µl)]
[RNase-free water]
?. [cDNA synthesis]
Incubate the mixture in a thermocycler for  at   followed by  at  and then  at  . Chill at .
23 °C
50 °C
80 °C
4 °C
?. [cDNA synthesis]
Dilute  cDNA in  RNase-free water for use as qPCR template. No dilution is required for use as a PCR template.
1 µl
14 µl
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]
Evaluate the level of isotopic enrichment using a qPCR assay. We recommend
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?. [Evaluate the level of enrichment]
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.
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
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)