Stable-Isotope Probing (SIP)

Introduction

Several features of RNA make it an excellent biomarker for use in stable-isotope probing (SIP) studies. RNA, having preceded the evolution of DNA and proteins, is one of the oldest molecules in life. It is common to all life and. RNA is turned over independently of cellular replication, and rapidly so in periods of activity. RNA is information-rich. A lot of tools and an enormous database to distinguish between types is available.

The RNA SIP protocol application led to the isolation and subsequent genome sequencing of the dominant phenol- degrading bacterium (Thauera sp) from an industrial wastewater treatment plant. In extension, RNA-SIP has been applied to assess the relationship between functional diversity and process stability in industrial wastewater treatment. RNA-SIP was also used successfully to identify and then isolate a bacterium that actively degrades benzene under denitrifying conditions in contaminated groundwater.

It has been applied to the identification of propionate oxidizers and methylotrophs in rice field soil, the identification of pentachlorophenol degraders in pristine grassland soil and the identification of bacterial micropredators in a soil trophic network. The most ambitious application of RNA-SIP has been in the identification of microbes in rhizosphere communities assimilating carbon from root exudates fixed by plants from 13C-labeled CO2.

Procedural notes

The protocols outlined here detail the formation,  ultracentrifugation and fractional analyses of gradients for the separation of 13C stable isotope-labeled RNAs extracted from the environment. In the majority of RNA-SIP applications, we extraction is the initial step, followed by selective purification of RNA (and DNA if required for DNA-SIP) using a commercially available kit. The combination of these protocols yields sufficient quality RNA for downstream processing in RNA-SIP investigations, and we have found them to be applicable for soil, water, bioreactor and litho-bio samples.

The protocol described here is optimized for 2.2 ml tubes centrifuged in a fixed-angle table-top ultracentrifuge rotor. For RNA-SIP with other (vertical) rotors and tubes (e.g. 5 ml), a straightforward adaptation of the presented protocol is required to other rotor geometries.  Finally, the initial publication of RNA-SIP3 relied upon reverse transcription–PCR of whole fractionated gradients followed by denaturant gradient gel electrophoresis analyses of each fraction to determine the migration points of labeled and unlabeled RNAs.

Materials & Reagents

  • Agarose
  • Tris-borate EDTA buffer
  • Ethidium bromide, 10 mg ml-1
  • Cesium trifluoracetate (CsTFA) 2.0 g ml-1
  • Nuclease-free water
  • Deionized formamide
  • Nuclease-free x 10 TrisEDTA
  • Isopropanol
  • Ethanol
  • Parafilm M
  • AccessQuick RT-PCR System
  • Bovine serum albumin, 20 mg/ml
  • SYBR Green I 10,000 X concentrate
  • Primers at 50 mM stock solution
  • 16S and 23S ribosomal RNA standard (from Escherichia coli), 100 mg/ ml in TE buffer, as contained in the RiboGreen RNA Quantitation Kit.

Equipment

  • Beckman TLX benchtop ultracentrifuge
  • Beckman tube topper
  • Beckman Fraction Recovery System
  • Razel syringe pump
  • GeneQuantPro RNA/DNA calculator
  • Certified DNA/RNase-free filter tips
  • Fully calibrated Gilson pipettes
  • 5 ml microfuge tubes
  • 0 ml microfuge tubes
  • 2 ml polyallomer sealable centrifuge tubes
  • 2 ml Plastipak syringe
  • 5 ml Plastipak syringe
  • 23 gauge Luer lock needles
  • Nitrile gloves
  • Three figure mg balance
  • Real-time PCR thermal cycler
  • UV sterilizing PCR workstation
  • Eight-strip PCR tubes and caps
  • Eight-channel pipettes

 Reagent Setup

CsTFA gradient with a starting density of 1.8 g/ml

For a 2.2 ml volume gradient, mix 1.761 ml of 2.0 g/ml CsTFA with 75 ml of deionized formamide and 344 ml nuclease-free water. This leaves 20 ml remaining for the RNA sample addition. If the volume of gradient medium is different from 2.2ml, the volumes can be scaled by taking into account the increased volume of the tubes relative to 2.2 ml. For multiple samples, make a ‘‘master mix’’ containing all the reagents needed for the number of gradients required and aliquot 2.180 ml of individual gradients from this stock before adding 20 ml of sample. This avoids gradient-to-gradient variation within a centrifugation batch due to pipetting errors.

20 X SYBR Green working solution

Dilute SYBR Green stock in a 1:500 volume ratio in nuclease-free water.  Diluted SYBR Green working solution is of limited stability. Store frozen (_20oC) in aliquots and thaw only once.

Procedure

13C-labeled substrate incubations and RNA extraction and purification

  1.  Pulse appropriate samples with 13C-labeled substrate at concentrations that are appropriate to the experimental question. It is advisable to perform initial investigations, which involve the 12C versions of substrates backed by chemical analyses to assess a time course for incorporation before applying the expensive 13C substrate. Once an incorporation rate is assessed for an environment, apply the 13C pulse, using chemical or isotopic ratio mass spectrometry-based analyses (the latter is highly desirable to specifically analyze 13C compound transfer). This allows a further assessment of incorporation rates and when compared to 12C compound kinetics, can be used to assess any degree of isotopic fractionation between 12C and 13C compounds. Samples obtained from the pulses can be stored at 20oC for up to 1 month or indefinitely at 70oC.
  2. Extract RNA or total nucleic acids according to a trusted protocol. If the latter (total nucleic acids) is obtained from the given protocol, further purify the nucleic acids to obtain a pure RNA preparation.
  3. Once RNA is purified and washed on the Qiagen column, elute in 50 ml of nuclease-free water and run 5 ml on a 1.5% (w/v) agarose gel containing 200 ng/ml ethidium bromide in 1X TBE at 70 V for 20 min. Visualize the gel and look for intact 16S and 23S rRNA. Once confirmed, determine the RNA concentration by spectrophotometry and dilute a portion of the RNA extract down to 100 ng/ml_with nuclease-free water.

RNA gradient preparation and centrifugation

  •   Form enough gradient medium, including formamide and excess water to allow an even number of centrifuge tubes, and including at least one blank gradient and control 12C RNA gradient. Blank gradients are included in a run as a reference gradient, the density profile of which is calculated after fractionation to assess the efficiency of ultracentrifugation. These should be performed for each centrifuge run. The 12C control gradients (samples pulsed with 12C substrates in parallel to the 13C pulse) are used for assessing the location of unlabeled RNA horizons, and for variation in banding density in unlabeled samples due to factors such as GC content.
  • For each sample, add 20 ml of RNA sample (containing approximately 500 ng of RNA; for example, 5 ml of 100 ng/ml sample from Step 4 added to 15 ml of nuclease-free water) to a clean 2 ml microfuge tube. For the blank gradient(s), add 20 ml of nuclease-free water. For 2.2 ml gradients, 500–600 ng of RNA is optimal. Adding more RNA at this stage can overload the gradient and distort its shape.
  • To the microfuge tube, add 2.180 ml of premixed gradient medium (a 2 ml microfuge tube will accommodate this without closing the lid). Withdraw the mixed medium and sample with a 2 ml syringe and 24-gauge needle and place into a polyallomer QuickSeal tube by placing the syringe needle in the open tube neck, tilt the tube and slowly fill the tube to avoid air bubbles.
  •  Seal the tube with a heat sealer (e.g., a Beckman ‘‘Tube Topper’’) and ensure the seal is complete and straight. Place the tubes in the rotor, note the tube position and place a shoulder cap on the tubes to support the tube tops and avoid tube crushing during centrifugation.
  • Spin at 128,000gav (64,000 r.p.m. in the TLA120.2 rotor) for 42–65 h at 20oC, with maximum acceleration and maximum deceleration. Owing to the density of CsTFA, rotors need to be de-rated to a maximum run speed equivalent to 80% of their normal maximum speed. Further, depending on rotor and tube configurations, the run times and speeds may vary. Vertical rotors are the most efficient at separation with correspondingly reduced run times. Fixed-angle rotors are the second choice, whereas swingout rotors are not compatible with forming such shallow isopycnic gradients. Conversion of our protocol’s centrifugation speed and duration to accommodate different rotor and centrifuge combinations can be obtained by using k-factors, based upon basic rotor dimensions, and can be achieved online.

Gradient fractionation

  1. Carefully remove the tubes from the centrifuge rotor with forceps and place in a rack.
  2. Prepare the top displacement gradient fractionator by connecting tubing into the fractionator hood to a 5 ml syringe filled with 5 ml of nuclease-free water. For ease, 5 ml of any DNA loading buffer can be added to the displacement water to give it coloration and allow visualization of the interface between the displacement water and CsTFA. Place the 5 ml syringe in the syringe pump and prime the line by pressurizing the syringe to force water through the line until a single drop emerges out of the fractionator hood. For optimum fractionation, use a fraction recovery system that allows fraction collection from the base of the tube via water displacement at the top of the tube. Controlled flow rates for displacement by water are obtained by coupling this system to a low flow rate syringe pump (capable of delivering 200 ml/min). Manual fractionation, especially for small volume gradients, is extremely difficult to control accurately.
  3.  Carefully place the centrifuge tube in the fraction recovery system and remove the top with a tube cutter. Lower the fraction recovery hood onto the top of the open tube neck and ensure a tight seal is obtained.
  4.  Pierce the bottom of the tube by inserting the fraction collection needle into the bottom of the tube and ensure a tight seal is maintained—all liquid should remain inside if a tight seal is formed. The seal can be enhanced by carefully stretching a small piece of Parafilm over the base of the tube.
  5.  Set the syringe pump to a flow rate of 200 ml/min, switch on the pump and start a stopwatch. Collect a fraction every 30 s, amounting to approximately 100 ml per fraction and 20 fractions per gradient. Other fractionation intervals can be chosen as required, especially for larger volume tubes. Collect fractions directly into nuclease-free 1.5 ml microfuge tubes. The whole process should take 10 min and result in approximately 20 fractions. Those that contain the displacement water toward the end of the fractionation will have a colored tinge owing to the loading buffer in the displacement water.
  6.  Calculate the absolute density and plot the gradient shape using density in g/ml as a function of fraction number, remembering that fraction 1 is the bottom of the gradient and fraction 20 is the top of the gradient. Generally, we suggest to fractionate the control (no RNA) gradient first and weigh 50–100 ml portions accurately on a three-figure balance (or alternatively, calculate the density by refractometry).
  7.  After each gradient is fractionated, clean the fractionator by removing the fractionator hood and pipetting 2 ml of 0.1 M NaOH into the empty centrifuge tube, allow to run out of the collection needle and repeat the process with 2 ml absolute ethanol.

RNA precipitation

  1.  To the 100 ml fractions, add two volumes (200 ml) of ice-cold isopropanol and incubate the tubes at 20oC for 30 min.
  2.  Centrifuge precipitations for 20 min at 14,000 g in a chilled microtube centrifuge at 4oC.
  3.  Remove the supernatant with a pipette and add a further 150 ml of ice-cold isopropanol. Care must be taken here, as, in general, RNA pellets will not be visible owing to the low loading capacity of the gradients. Further, efficient washing with isopropanol is required to remove the CsTFA, which inhibits downstream enzyme reactions.
  4. Spin at 14,000 g for 5 min at 4oC and remove the supernatant. Spin the tube for the final time for 1 min at 14,000 g and remove any excess isopropanol using a 20 ml volume pipette and tip. This ensures a short period for sample drying.
  5.  Air-dry samples and resuspend dried pellets in 10 ml of RNase-free TE.

RT-PCR quantification of bacterial rRNA in gradient fractions

  1.  Prepare sufficient master mix for qRT-PCRs (40 ml each) in a nuclease-free 2 ml microfuge tube: Nuclease-free water :16.4 ml, AccessQuick 2X master mix: 20 ml: 1X conc., BSA (20 mg/ml): 0.4 ml: 0.2 mg/ml conc., SYBR Green working solution (20X): 0.2 ml: 0.1x conc., 519f-primer18 (50 mM): 0.2 ml: 0.25 mM conc., 907r-primer19 (50 mM): 0.2 ml: 0.25 mM conc, AMV (5 U/ml): 0.6 ml: 3 U conc. This protocol utilizes short amplicons generated with universal primers targeting bacterial 16S rRNA, but other laboratory-specific primers generating up to 500 bp PCR products can be used, as long as they have homology to the E. coli standard rRNA. Take maximum care to avoid contamination of the qRT-PCR master mix and reactions during set up. The reaction is extremely sensitive to the carryover of bacterial SSU rRNA PCR amplicons from previous experiments. Work within a UV-cabinet and use only UV-sterilized plastic ware, if possible.
  2.  Pipette 5 ml of E. coli standard rRNA (100 ng/ml) into the first tube of an eight-tube PCR strip. Dilute 1:10 by adding 45 ml of nuclease-free water and mix by pipetting 1–2 times.
  3.  Pipette 5 ml of E. coli standard rRNA from first tube (10 ng/ml) into the second tube of the eight-tube PCR strip. Dilute 1:10 by adding 45 ml of nuclease-free water and mix by pipetting 1–2 times. Continue to dilute standard in 10-fold steps until tube 8 (10-6 ng/ml). E. coli standard rRNA 10-fold dilution series are extremely unstable and cannot be stored for more than 1 h at 4oC or frozen. They must be freshly prepared for each qRT-PCR experiment.
  4.  Pipette 2 ml of each gradient fraction rRNA sample (unknowns) into adjacent tubes within eight-tube PCR strips. Three strips are required for each gradient.
  5.  Add 38 ml of qRT-PCR master mix (Step 22) to each template and mix by pipetting 1-2 times. Seal eight-tube PCR strips with appropriate caps.
  6.  Pipette 2 ml of nuclease-free water (no-template controls) into several (2–4) replicate tubes of a new eight-tube PCR strip. Continue as in Step 25.
  7.  Pipette 2 ml of each E. coli rRNA dilution (standards) into adjacent tubes of an eight-tube PCR strip. Replicate over multiple eight-tube strips as required. Continue as in Step 25. To stabilize dilute rRNA during setup, all samples and reactions must be continuously kept at 4oC or in an ice bath. To decrease the total handling time, pipetting should be conducted with eight-channel pipettes.
  8.  Place the eight-tube PCR strips into an appropriately programmed real-time PCR thermal cycler and amplify using the following durations and temperatures. Collect SYBR Green fluorescence data for each reaction in steps as indicated below.

 

Step                                       Temperature (oC)   Duration       Fluorescence

Reverse transcription          45                                  20 min

Initial denaturation               95                                  5 min

35 cycles of:

Denaturation                         95                                  30 s

Annealing                              52                                  30 s              SYBR

Elongation                             68                                  30 s

Final elongation                   68                                  5 min

Final denaturation               95                                  1 min

Reassociation                       55                                  30 s

Dissociation ramp                55–95                           30 min                      SYBR

Final hold                              25                                  Hold

Quantify bacterial rRNA in each gradient fraction to arbitrary E. coli 16S and 23S rRNA (ng/ml) units via the measured SYBR Green fluorescence threshold cycles (Ct) in each qRT-PCR reaction. Care must be taken to omit from analyses false-positive Ct values that may be caused by the formation of primer dimers in samples containing no or extremely low amounts of template rRNA. These can be identified by the melting profiles of PCR products recorded during the dissociation ramp. This standard procedures and instrument setup are not explained here in detail but can be found in every qPCR instrument manual.

Timing

To set up ten gradients it takes around 1 h and after centrifugation, RNA gradient fractionation of ten gradients takes around 2.5 h. Subsequent qRT-PCR of 2–4 fractionated rRNA centrifugation gradients are usually set up within 1–1.5 h and run within 2.5h.