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

[DESCRIPTION]
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.
 

 

 


[BEFORE_START]
Prepare all buffers and solutions in advance (see Step 1).
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.

 


[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).
 

 

 

 

 


[STEPS]
SECTION: Solutions for SIP
1. Prepare the following solutions:

 

SECTION: Solutions for SIP
1.1. Gradient buffer (0.1 M Tris-HCl, 0.1 M KCl, 1 mM EDTA) pH 8.0 :
15.76 g 
7.455 g 
0.37224 g 

Dissolve the salts in RNase-free water and fill up to1000 ml. Filter sterilise (0.1-0.2 μm). Autoclave.
 

 

 
Store atRoom temperature 
SECTION: Solutions for SIP
1.2. Molecular-grade ethanol solution (75 % (v/v)):
75 ml 
25 ml 
Store at -20 °C 



SECTION: Solutions for SIP
1.3. If preparing CsTFA solution from powder:
Pour17.2 ml directly into a50 g vial.
Mix by hand atRoom temperatureand then allow for the residues to fully dissolve during several hours or overnight.
If particle impurities are visible, filter the solution into a fresh bottle using a 0.1 or 0.2 μm filter.
Store at4 °C. 

 


  

SECTION: Gradient preparation
2. Calibrate the refractometer with RNAse-free water at20 °C .
30 µL
Following calibration, the device should read 1.3330 ± 0.0002 nD-TC 

 

SECTION: Gradient preparation
3. For every two gradients, mix the following in a 50 ml tube (assuming 6 ml Ultracrimp, PA centrifugation tubes): 
9.696 ml
2.166 ml
65 Room temperature 
 

  

 


SECTION: Gradient preparation
4. Mix by inverting several times, pipette30 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.

SECTION: Gradient preparation
5. Add3.56 % (v/v)  HiDi (422 µL if the volume was not corrected).

 

SECTION: Gradient preparation
6. Measure the density.  Make sure the density is: 1.3725 ± 0.0002 nD-TC.

 

SECTION: Gradient preparation
7. Transfer ca.5.8 ml of the mixture to each centrifugation tube using a micropipette. Make sure the volume reaches only the bottom of the neck.

SECTION: Gradient preparation
8. 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
150 ng/µl 

 

SECTION: Gradient preparation
9. 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.
SECTION: Gradient preparation
10. Close the caps (using an appropriate crimper or by hand, depending on the type of tubes).
 

SECTION: Gradient preparation
11. 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.
SECTION: Ultracentrifugation
12. Centrifuge 
130000 x g, 3900 min, 20 °C 

Maximum acceleration and deceleration.
 

SECTION: Fractionation
13. Prepare a rack filled with 2.0 ml low-binding collection tubes (one per fraction).
 

SECTION: Fractionation
14. Fill a 20 ml syringe with RNase-free water. Remove any air bubbles. 
SECTION: Fractionation
15. 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.
 

 

 

 

 

SECTION: Fractionation
16. Set the Rate to 1 ml min-1 and collect fractions in30 s steps. If using a 6 ml tube, this will yield 12 fractions. Volume should be set to "off" and diameter to "22 mm".

 

 

SECTION: Fractionation
17. 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.
SECTION: Fractionation
18. 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.

 

SECTION: Fractionation
19.  Pierce the ultracentrifugation tube, just below the neck, using the needle attached to to the precision pump tube.
  

 

SECTION: Fractionation
20. 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.

 

SECTION: Fractionation
21. 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:
  

SECTION: Fractionation
22. Start the pump, as soon as the first drop falls off the ultracentrifugation tube start the stopwatch
SECTION: Fractionation
23. After 30 s (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.

SECTION: Fractionation
24. 
Measure the density of each fraction using the refractometer. Start from the last (the lightest) fraction.
30 µL 
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).

  
 


SECTION: RNA precipitation
25. 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 µLfractions were collected and 30 µL  were spent for determining the density, add 47 µL Na-acetate and 1175 µL. 
 

 

 


SECTION: RNA precipitation
26. Incubate at -80 °C  for 30 min.

SECTION: RNA precipitation
27. Centrifuge at 14000 rpm, 30 min, 4 °C.

SECTION: RNA precipitation
28. Decant the supernatant, wash once with 1 ml, invert the tube several times. 

 



SECTION: RNA precipitation
29. Centrifuge at 14000 rpm, 10 min, 4 °C.

SECTION: RNA precipitation
30. 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. 
 

SECTION: RNA precipitation
31. 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.
5 min

 

SECTION: RNA precipitation
32. Resuspend the pellets in 10 µL RNase-free water or the RNA Storage solution.
 

SECTION: cDNA synthesis
33. For each fraction, prepare the following mixture in a PCR tube:
10 µL
3 µL(50 micromolar (µM) diluted 20x in RNase-free water: 2.5 micromolar (µM))

 

SECTION: cDNA synthesis
34. Incubate the mixture at 65 °C for 5 min in a thermocycler and chill at 4 °C  for at least 1 min.

SECTION: cDNA synthesis
35. Prepare the following mixture (times the number of fractions) and add 7 µL into each tube:

4 µL 
1 µL 
1 µL 
0.2 µL 
0.2 µL 
0.1 µL 
0.5 µL 
 

 

 

 

 

SECTION: cDNA synthesis
36. Incubate the mixture in a thermocycler for 10 min at 23 °C  followed by 60 min at 50 °C and then 10 min at 80 °C . Chill at 4 °C.
SECTION: cDNA synthesis
37. 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. 
 


SECTION: Evaluate the level of enrichment
38. 
Evaluate the level of isotopic enrichment using a qPCR assay. We recommend 

 

SECTION: Evaluate the level of enrichment
39. 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.