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Dec 2020

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RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities
RI-SEC-seq:具有不同稳定性的非泡性细胞外RNA的综合分析   

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Abstract

Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and remain in ultracentrifugation supernatants of cell-conditioned medium or blood serum. We have observed that nonvesicular exRNA profiles are highly biased toward those RNAs with intrinsic resistance to extracellular ribonucleases. These highly resistant exRNAs are interesting from a biomarker point of view, but are not representative of the actual bulk of RNAs released to the extracellular space. In order to understand exRNA dynamics and capture both stable and unstable RNAs, we developed a method based on size-exclusion chromatography (SEC) fractionation of RNase inhibitor (RI)-treated cell-conditioned medium (RI-SEC-seq). This method has allowed us to identify and study extracellular ribosomes and tRNAs, and offers a dynamical view of the extracellular RNAome which can impact biomarker discovery in the near future.


Graphical abstract:

Overview of the RI-SEC-seq protocol: sequencing of size-exclusion chromatography fractions from nonvesicular extracellular samples treated or not with RNase inhibitors (+/- RI)



Keywords: exRNA (细胞外RNA), RNA biomarker (RNA生物标志物), Intercellular communication (细胞间通讯), Next generation sequencing (新一代测序), Exosomes (外泌体), Liquid biopsies (液体活检), Differential stability (不同稳定性), Extracellular ribosomes (细胞外的核糖体)

Background

Extracellular RNAs (exRNAs) are involved in intercellular communication and are promising disease biomarkers in minimally invasive liquid biopsies (O’Brien et al., 2020). To be transported in human bodily fluids, exRNAs need to achieve some degree of protection against extracellular ribonucleases, which are ubiquitous in the extracellular milieu. By far, the most studied and best understood mechanism is RNA encapsulation inside extracellular vesicles (EVs) and release of these endogenous lipid nanoparticles into the extracellular space (Kalluri and LeBleu, 2020). Beyond simply protecting exRNAs from degradation, EVs contain a set of surface proteins which are involved in cellular uptake (Hoshino et al., 2015), behaving as vehicles for RNA transfer between cells. This can occur both physiologically (Thomou et al., 2017) or under therapeutic biotechnological setups (Kamerkar et al., 2017; Kojima et al., 2018).


Much less is known about those exRNAs which are not associated with EVs (Li et al., 2018). However, early work has shown that the majority of microRNAs in human plasma are forming soluble ribonucleoprotein complexes with Argonaute 2 and are not transported inside EVs (Arroyo et al., 2011; Turchinovich et al., 2011). Later work has shown that the RNA content of EVs is not as high as previously suggested (Chevillet et al., 2014) and we have observed that most exRNAs in cell culture media remain in ultracentrifugation supernatants (Tosar et al., 2015).


We were surprised to find that the majority of the small RNAs that we could sequence in ultracentrifugation supernatants of cell-conditioned medium were 5’ tRNA halves derived from glycine and glutamic acid issoaceptors (Tosar et al., 2015). These sequences were also the most abundant in blood plasma, serum and other biofluids (Srinivasan et al., 2019), where they also seemed to be transported outside EVs (Dhahbi et al., 2013). We later observed these sequences could form dimers and that these dimers were highly resistant to single-stranded ribonucleases (Tosar et al., 2018). These observations suggested that extracellular nonvesicular RNA profiles are prone to survivorship bias (Tosar et al., 2021), where we tend to see only those RNAs that are more resistant to degradation. Although this could be desirable for biomarker studies, it can be problematic for understanding RNA release mechanisms. Thus, we developed a method to study nonvesicular exRNAs irrespectively of their extracellular stability. This method, called RI-SEC-seq (for: size-exclusion chromatography [SEC] fractionation of RNase inhibitor [RI]-treated cell-conditioned medium), enabled us to study RNAs in the form they were effectively released from cells. Following this procedure, we discovered the presence of extracellular ribosomes and tRNAs, and the extracellular biogenesis of extracellular tRNA-derived fragments (Tosar et al., 2020). The latter was independently validated by others, using cell lines devoid of RNase I (Nechooshtan et al., 2020).


Here, we provide the experimental procedure so that readers can also obtain snapshots of the extracellular nonvesicular RNAome under low, intermediate and high RNase activities. This will help to characterize exRNA profiles in a comprehensive and dynamic manner, and to identify other stable RNAs with biomarker potential.


Materials and Reagents

  1. T-75 flask (Corning, catalog number: 430641U)

  2. 0.22 µm or 0.45 µm membrane (Millipore, catalog number: GSWP04700)

  3. MCF-7 cell line (ATCC, ATCC® HTB-22TM), storage: liquid nitrogen when not in use. Cryopreservation: follow ATCC recommendations

  4. 1 ml syringe

  5. 14 ml, Open-Top Thinwall Ultra-Clear Tube, 14 × 95 mm, 50Pk (Beckman Coulter Life Sciences, catalog number: 344060), storage: room temperature

  6. Vivaspin 20 centrifugal concentrator, MWCO 10 kDa, PES membrane (Sartorius, catalog number: VS2001), storage: room temperature

  7. 96 Well Standard Assay Block, 2 ml, Certified RNase/DNase free, for collection of chromatographic fraction (Costar, Corning Incorporated, catalog number: 3961), storage: room temperature

  8. Superdex® 200 10/300 GL (GE Life Sciences, Sigma/Merck, catalog number: GE17-5175-01, discontinued product; alternative: Superdex® 200 Increase 10/300 GL, catalog number: GE28-9909-44), storage: room temperature

  9. DMEM, high glucose, GlutaMAX TM Supplement (Gibco, Thermo Scientific, catalog number: 10569010), storage: 4 °C

  10. Fetal bovine serum (FBS), qualified, origin: Brazil (Gibco, Thermo Scientific, catalog number: 12657029), storage: aliquoted (sterile) at -20 °C

  11. MEGMTM Mammary Epithelial Cell Growth Medium BulletKitTM (Lonza, catalog number: CC-3150), storage: the basal MEBM medium is stored at 4 °C

    Note: The MEGM SingleQuotsTM Supplements are stored at -20 °C. Once mixed, the complete MEGM medium is stored at 4 °C until expiry. We do not add the bovine pituitary extract or the antibiotics included in the kit.

  12. DPBS, no calcium, no magnesium (Gibco, Thermo Scientific, catalog number: 14190144), storage: 4 °C. Use this sterile solution for cell culture

  13. DPBS (10×), no calcium, no magnesium (Gibco, Thermo Scientific, catalog number: 14200075). Make 1× solution with ultrapure or DEPC (Diethyl pyrocarbonate)-treated water, filter, and use as mobile phase in size-exclusion chromatography

  14. Trypsin-EDTA (0.05%), phenol red (Gibco, Thermo Scientific, catalog number: 25300062), storage: -20 °C

  15. RNase inhibitor, murine (New England Biolabs, catalog number: M0314S), storage: -20 °C

  16. TRIzolTM reagent (Invitrogen, Thermo Scientific, catalog number: 15596018), storage: 4 °C

  17. TRIzolTM LS reagent (Invitrogen, ThermoScientific, catalog number: 10296028), storage: 4 °C

  18. 2-propanol, for molecular biology (Sigma-Aldrich, catalog number: I9516-500ML), storage: room temperature. Reagent needed for RNA purification based on the TRIzol method

  19. Ethyl alcohol, pure, for molecular biology, storage: room temperature. Reagent needed for RNA purification based on the TRIzol method

  20. Glycogen, RNA grade (Thermo Scientific, catalog number: R0551), storage: -20 °C. Reagent needed for RNA purification based on the TRIzol method

  21. UltraPureTM DNase/RNase-Free Distilled Water (Invitrogen, ThermoScientific, catalog number: 10977035), storage: room temperature, sterile

    Note: We do not use this water for large volumes of buffers such as those used in size-exclusion chromatography. For those purposes, we use MilliQ water that we validate as RNase-free in house. Autoclaved DEPC-treated water can be used instead. In this case, add DEPC (diethylpyrocarbonate, Sigma, catalog number: D5758) to a final concentration of 0.1% v/v, incubate overnight at room temperature and then autoclave to inactivate DEPC.

  22. 10× Tris-Borate-EDTA Electrophoresis Buffer (ThermoScientific, catalog number: B52), storage: room temperature

  23. Acrylamide/bis-Acrylamide 30% solution, bioreagent, suitable for electrophoresis, 29:1 (Sigma-Aldrich, catalog number: A3574), storage: 4 °C. Hazardous (read MSDS)

  24. SYBRTM Gold nucleic acid gel stain (10,000× concentrate in DMSO) (Invitrogen, ThermoScientific, catalog number: S11494), storage: -20 °C

  25. RNA loading dye, 2× (New England Biolabs, catalog number: B0363S), storage: -20 °C

  26. QubitTM RNA HS Assay Kit (Invitrogen, ThermoScientific, catalog number: Q32852), storage: 4 °C

  27. NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, catalog number: E7300S), storage: -20 °C

  28. MiSeq Reagent Kit v2 (300-cycles) (Ilumina, catalog number: MS-102-2002), storage: -20 °C (box 1) and 4 °C (box 2)

Equipment

  1. NovexTM TBE-Urea Gels (ThermoFisher, catalog number: EC6875BOX)

  2. XCell SureLock Mini-Cell (ThermoFisher, catalog number: EI0001)

  3. ÄKTA pure FPLC system (Cytiva, former GE Life Sciences) with multiple wavelength detectors (at least capable of simultaneous detection at 280 and 260 nm) and an automatic fraction collector with capacity for 96 well assay blocks (Costar, 2 mL, RNase-free, catalog number: 3961)

  4. QubitTM Fluorometer 2.0 (Thermo Scientific, catalog number: Q32866, discontinued product. Replaced by Catalog number Q33216)

  5. MiSeq Next Generation Sequencer (Illumina)

Software

Note: All software used for sequencing data analysis was run using the Galaxy Europe web interface (https://usegalaxy.eu).

  1. fastx_clipper (fastx_toolkit)

  2. Fastqc

  3. RNA STAR

  4. BAM filter

  5. bamCoverage

  6. featureCounts

Procedure

Note: The chances of RNase contamination should be minimized throughout this protocol even if not specifically indicated. Use disposable RNase-free plastics and certified RNase-free solutions whenever possible. Use gloves in all steps even when handling non-hazardous reagents and work in RNase-decontaminated surfaces with RNase-decontaminated materials.


  1. Cell culture

    Note: For this procedure to be successful, cells must be grown in serum-free medium (SFM), to avoid the presence of serum RNases. In this protocol, we will use Mammary Epithelial Growth Medium (MEGM, Lonza) and MCF-7 cells, but this can be adapted to other cell lines. However, finding a suitable SFM for each cell line is not trivial. Serum deprivation can induce cell stress and this can affect both intracellular and extracellular RNA profiles. As described in Tosar et al. (2020), we monitor this by Western blot, looking at eIF2 subunit α (Ser 51) phosphorylation levels, and by nonradioactive Northern blot, monitoring the levels of stress-induced tRNA-derived fragments (Yamasaki et al., 2009). For certain cell types, commercial SFM formulations are available. We have observed that MCF-7 cells can be grown for short periods of time in MEGM, which is the recommended growth medium by ATCC for other mammary epithelial cell lines such as MCF-10A. However, we do not add the Bovine Pitutary Extract contained in the MEGM kit to keep this as a chemically-defined medium. We also do not add antibiotics to our cell cultures unless needed. In theory, this protocol could be applied to suspension cells, 3D cultures and organoids with slight modifications, although we have not tried these applications yet.

    1. Seed aprox. One million cells in each of two T-75 flask. Grow cells up to 60-70% confluency in complete medium (e.g., DMEM + 10% FBS) at 37 °C and 5% CO2. Confluency should be adapted depending on the growth rate of each cell line. The aim is 80-90% confluency 48 h later.

    2. Wash cells briefly with 3-5 ml pre-warmed DMEM or PBS.

    3. Add 10 ml of pre-warmed MEGM. Grow cells for 24 h.

    4. Wash cells again with pre-warmed MEGM. Add 10 ml of pre-warmed MEGM to one of the flasks (hereafter: control). Add 10 ml of pre-warmed MEGM + 80U (2 µl) of murine RNase inhibitor to the second flask (hereafter: RI-treated) and incubate for further 24 h.

    5. Aspirate cell-conditioned medium from both flasks and discharge in 15 ml nuclease-free Falcon conical tubes. Add 1 µl of murine RNase inhibitor to the RI-treated flask (only). Spin down cells at 800 × g for 5 min at room temperature.

    6. While centrifuging cell-conditioned medium, move the empty flasks containing the cell monolayers to a fume hood and lyse cells in 2 ml of Trizol. Gently tilt the flasks to lyse all cells, incubate 5 min at room temperature, close the lid and store the flask at -20 °C. This will be later used to obtain intracellular RNA profiles.

    7. Place the supernatant from Step A5 to a new 15 ml nuclease-free Falcon conical tube and centrifuge at 2,000 × g for 20 min at 4 °C. We routinely store the supernatant from this spin (saved in a new tube) at -20 °C for later use.


  2. Preparation of extracellular nonvesicular samples by ultracentrifugation

    Note: Ultracentrifugation requires specific training in situ. This protocol is not intended to be substitute for this training. Improper use of ultracentrifuges can provoke sample lost, permanent damage to expensive equipment and put the safety of the laboratory personnel at risk.

    1. Thaw the tubes from Step A7 by placing the tubes in a beaker containing water at room temperature. Exchange the water several times, as it will cool rapidly.

    2. Add 1 µl of murine RNase inhibitor to the tube containing the RI-treated sample. Because recombinant RNase inhibitors contain many cysteines and are prone to oxidation, we usually add this boosters to ensure lack of RNase activity.

    3. Spin the samples again at 2,000 × g for 20 min at 4 °C to remove precipitates that could have formed during freeze/thaw.

    4. Place the samples in two identical ultraclear ultracentrifugation tubes. If necessary, adjust the volume of the samples with ice-cold nuclease-free PBS so that the tubes are up to their full capacity minus 2-3 mm from the top of the tube. All opposing tubes for a run must be filled to the same level with liquid of the same density.

    5. Place the tubes in the buckets. We also check that buckets containing filled tubes show a difference in weight equal or less than 2 mg if they were placed in opposite positions of the rotor. Place the buckets in an already cooled rotor (stored in a cold room overnight). Place the rotor in the ultracentrifuge, set all relevant parameters and start vaccum.

      Note: Forgetting to cool the rotors overnight played an important role in the development of this protocol. A science journalist wrote a story about this episode that gives some historical context (Khamsi, 2020).

    6. Run for 2.5 h at 100,000 × g at 4 °C with maximum acceleration and break.

    7. Transfer the supernatants from Step B6 to new tubes. Keep the tubes on ice.

    8. Concentrate to 500 µl by ultrafiltration at 4 °C, using Vivaspin 20 units with a molecular weight cut-off of 10 kDa. Run ultrafiltration units with nuclease free PBS or water before loading the samples in order to clean the membranes. Transfer the concentrate to a new, nuclease-free 1.5 ml tube.


  3. Size-exclusion chromatography

    Note: This procedure needs to be done twice, with the RI-treated and control samples. Working with an FPLC system also requires specific training in situ as high pressures will be used. Avoid introduction of air to the column at all stages and the injection of samples or solvents containing dust or other particulates.

    1. Filter 500 ml of ultrapure water and 500 ml of 1× PBS (prepared in ultrapure water) through a 0.22 µm or a 0.45 µm membrane using a vacuum filtration device. Degass the solutions with a magnetic stirrer under reduced pressure.

    2. Set an alarm in the FPLC system lower or equal to the maximum pressure admitted by the column (in this case, 1.5 MPa). Connect a Superdex 200 10/300 column to the FPLC system. Remove ethanol from the column by passing at least 1.5 volumes of filtered and degassed water. Equilibrate the column in 1× PBS.

    3. Connect a 500 µl loop to the system. Clean the loop with water, and then with filtered PBS. Avoid the introduction of air bubbles.

    4. Spin the sample (from Step B8) at 10,000 × g for 10 min at 4 °C. Transfer the supernatant to a new tube.

    5. Using a 1 ml syringe, inject the sample from Step C4 (500 µl) in the column.

    6. Run at a flow rate of 1 ml/min by monitoring absorbance at 280 nm and 260 nm, simultaneously.

    7. Collect fractions of 0.25 ml using a new, RNase-free 96-well plate. Start collection at least 2 ml before elution of the fractions corresponding to the void volume.

    8. Identify fractions were ABS260 > ABS280 in both samples. In contrast to proteins, nucleic acids absorb more UV light at 260 nm than at 280 nm. Thus, these fractions correspond to the elution of nucleic acids or ribonucleoprotein complexes. At least in MCF-7 cells, the control sample should show a main 260 nm peak at Ve = 15 ml (that we call “P1”: adjusted Ve’ = 7.5 ml) and a lower peak at Ve = 16.5 ml (that we call “P2”: adjusted Ve’ = 9.0 ml). In contrast, the P2 peak should not be seen in the RI-treated sample, and a new peak should appear in the void volume (that we call “P0”: Ve = 7.5 ml; adjusted Ve’ = 0 ml) (Figure 1).



      Figure 1. Representative chromatograms of RI-treated (top) and control (bottom) samples. The absorbance at 260 nm and 280 nm is shown in red and blue, respectively. Peaks with 280/260 > 1 correspond to the elution of proteins, while peaks with 280/260 < 1 correspond to the elution of nucleic acids and/or ribonucleoprotein complexes. Ve: elution volume. Modified from Tosar et al. (2020).


    9. Transfer fractions corresponding to peaks P0, P1 and P2 from both samples to new 1.5 ml RNase-free tubes. Make sure that paired fractions correspond to the same elution volumes, even if no evident peaks are present (e.g., P0 in control or P2 in RI-treated).

    10. In a fume hood, add 750 µl of Trizol LS to each collected fraction (250 µl). Proceed with RNA extraction following the instructions of the manufacturers.


  4. RNA analysis and next-generation sequencing

    1. Resuspend the RNA pellet in 9 µl of RNase-free water. Check RNA concentration using 1 µl of each sample and a Qubit fluorimeter (following the instructions of the vendor). If using a Bioanalyzer to check RNA integrity, rembember these are not intracellular samples and therefore the concept of RNA Integrity Number (RIN) does not apply here. Nevertheless, the P0 peak in the RI-treated samples should contain intact ribosomal RNAs. Instead of using the Bioanalyzer, we characterize the RNAs in each fraction by denaturing gel electrophoresis using custom-made 6% polyacrylamide-8 M urea gels (P0) or 10% polyacrylamide-8M urea gels (P1 and P2) in 0.5× Tris-Borate-EDTA buffer. Mix 1 µl of each sample with 1 µl of a 2× loading dye suitable for denaturing polyacrylamide gel electrophoresis (Figure 2). Pre-cast gels can be used as a time-effective alternative to custom-made gels (e.g., Novex TBE-Urea, ThermoFisher, catalog number: EC6875BOX; run on the XCell SureLock Mini-Cell, ThermoFisher, catalog number: EI0001).



      Figure 2. Analysis of the P1 peak either with (+) or without (−) RI treatment (left gel) or the P0 peak (right gel) in a denaturing polyacrylamide gel. Cells: intracellular RNA extracted from MCF-7 cells. Bands corresponding to tRNAs, 5S rRNA, 5.8S rRNA, 7SL RNA, 18S rRNA and 28S rRNA are indicated. Numbers at the left (100, 200, 300) correspond to the migration of a RNA ladder (not shown). The gel shows that RNAs in the P1 peak are shifted from full-length tRNAs (+RI) to small RNAs of around 30 nt (-RI). In contrast, RNAs in P0 are considerably larger, and bands of the size of 28S, 18S and 5.8S rRNAs are evident. Modified from Tosar et al. (2020).


    2. Using the remaining 7 µl of each sample, proceed to sequencing library preparation using a small RNA kit such as the NebNext Small RNA sequencing kit for Illumina. Perform single-end sequencing in an Illumina platform. In our study (Tosar et al., 2020) we sequenced for 200 cycles in order to be able to detect some full-length RNAs (e.g., 5.8S rRNA) and not only their fragments, based on gel electrophoresis results (Figure 2). However, doing so has a major drawback. Since most of the clusters in the chip contain inserts of small size, sequencing for longer than 150 cycles provokes sequencing the entire adapter, polymerase run off, and abortive termination of the sequencing reaction before proceeding to demultiplexation, at least in a MiSeq benchtop sequencer as the one we used. Data analysis can still be performed because the index will be contained as part of the sequence of each read. However, extracting data from an aborted run is not straightforward, and we recommend not to proceed for longer than 100-150 cycles unless understanding exactly what is being done.

Data analysis

Analyze data as described in Tosar et al. (2020). Briefly, identify and clip 3’ adapter sequences, map clipped reads to the human genome and count features corresponding to microRNAs, tRNAs, rRNAs, snRNAs, snoRNAs, YRNAs and Vault RNAs. Low-quality reads can be eliminated, but do not trim low quality bases at the end of the reads because doing so generates artificial 3’ variants that complicate interpretation. A comparison of the P1 peak between RI-treated and control samples is shown in Figure 3. Note the identification of a putative dimer between an internal fragment of the 28S rRNA and the full-length U49 snoRNA in the RI-treated sample, which is completely absent in the control. This is an example of how RI-SEC-seq enables to identify extracellular nonvesicular RNA complexes that are lost in conventional exRNA profiling due to their low extracellular stability (compared to highly stable dimers formed by glycine 5’ tRNA halves of exactly 30-31 nucleotides) (Tosar et al., 2018 and 2020).



Figure 3. overview of RI-SEC-seq results. A. Size distribution of small RNA sequencing reads mapping to different ncRNAs (see legend) in the P1 peak of RI-treated (top) or control sample (bottom). RPM: reads per million mapped reads. Numbers indicate the length of 5’ tRNA-derived reads. B. Same, but showing only the reads aligning to rRNAs (28S rRNA in red, 5.8S rRNA in orange, 5S in black). In this case, numbers indicate starting position of most reads. C. Representation of the predicted SNORD49A (U49A; black)/28S rRNA (red) interaction, as depicted in snoRNABase (www-snorna.biotoul.fr). Below is the sequence with the highest number of reads. Its relative abundance is expressed as reads per million mapped reads (RPM). Its ranking in the “P1 + RI” dataset is also shown. Modified from Tosar et al. (2020).


Notes

  1. RI-SEC-seq incorporates the concept of differential extracellular stability in exRNA profiling. However, it suffers from the limitations of the sequencing method that is applied. For instance, we know that the P1 peak in RI-treated samples contains mainly full-length tRNAs, and this is evident in Figure 2. However, sequencing of P1 in RI-treated samples did not show a significant number of reads corresponding to full-length tRNAs. On the contrary, most of the tRNA-derived reads corresponded to 5’ tRNA halves (Figure 3). Thus, RI-SEC-seq should be combined with orthogonal techniques such as Northern blot and density gradient centrifugation to get a more representative picture of the extracellular RNAome. Alternatively, the small RNA sequencing technique described here can be substituted for alternative methods that are able to sequence both full-length tRNAs and their fragments, such as TIGRT-seq (Qin et al., 2016; Shurtleff et al., 2017).

  2. There is a change in the length of tRNA-derived fragments between P1 + RI and P1 – RI. Fragments of 33-34 nt are observed in RI-treated samples but they are lost in the controls (Figure 3A). A similar pattern is observed by Northern blot (Tosar et al., 2020). The reason is that the main cleavage products of samples containing full-length tRNAs (like PI + RI) are 5’ tRNA halves of 33-35 nt (and their cognate 3’ halves, usually harder to sequence). However, shorter 5’ halves of 30-31 tend to be much more stable. This could be due to the formation of dimers (Tosar et al., 2018) or interactions with extracellular RNA-binding proteins with size-dependent preferences. As a consequence, 5’ halves of 30-31 nt (mainly derived from tRNAGly) tend to accumulate when RNase activities are higher. Thus, RI-SEC-seq can identify variations in the stability of different RNAs within a single RNA class (e.g., between shorter and longer 5’ tRNA halves) as well as between different RNA classes (Figure 3A).

  3. Small RNA sequencing in P0 (RI-treated samples) showed a variety of rRNA-derived fragments, mainly derived from the 28S rRNA. However, Figure 2 clearly shows that the RNAs eluting in P0 have a high molecular weight. It should be noted that when applying small RNA sequencing to samples containing mainly full-length long non-coding RNAs (such as 28S and 18S rRNAs), only their fragments will be retrieved. This can lead to the misconception that exRNAs are mainly fragmented in situations where they are clearly not. This is further supported by our finding of a significant number of reads corresponding to the full-length 5.8S rRNA, which is sufficiently short to fit our sequencing window. By combining RI-SEQ-seq with mass spectrometry, Western blot and velocity gradients, we showed that the P0 peak contains ribonucleoprotein complexes composed of rRNAs and ribosomal proteins. Thus, development of the RI-SEQ-seq method was a key step leading to the discovery of extracellular ribosomes (Khamsi, 2020; Tosar et al., 2020).

  4. Applications of this protocol extend to any situation where it is important to capture the whole set of RNAs released to the extracellular space, rather than only the most stable ones (i.e., glycine or glutamic acid 5’ tRNA halves and EV-associated RNAs). For instance, testing how intracellular changes in gene expression between infected and noninfected cells, or between stressed and nonstressed cells, are reflected in the extracellular space. Additionally, RI-SEC-seq is a valuable tool to dissect extracellular RNA processing mechanisms and exRNA biogenesis.

Recipes

Commercial reagents and concentrated buffers were used through this protocol, as indicated above. Nevertheless, most of these buffers are routinely used in molecular and cellular biology laboratories and can be easily made from common solid stocks. In this particular case, we have opted to purchase commercial solutions to minimize RNase contamination throughout the protocol.

Acknowledgments

This work received funding from the following sources: Agencia Nacional de Investigación e Innovación (ANII, Uruguay) [FCE_3_2018_1_148745]; Comisión Sectorial de Investigación Científica (CSIC-UdelaR, Uruguay) [MIA_PAS2019_2_99] and National Institutes of Health, USA [UG3CA241694, supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director]. J.P.T. and A.C. are researchers from PEDECIBA (Uruguay), The National System of Researchers (ANII) and the full-dedication program of UdelaR.

    The rationale for this protocol was first presented in Tosar et al. (2018), but RI-SEC-seq was formally developed in Tosar et al. (2020). All figures shown in this protocol were adapted from the latter publication.

Competing interests

None declared

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简介

[摘要]外来体和其他细胞外囊泡(EVs)被认为是在细胞外样品(包括人体液)中运输RNA的主要载体。但是,大部分细胞外RNA(exRNA )不能与EV共纯化,而是保留在细胞条件培养基或血清的超速离心上清液中。我们已经观察到非囊泡的exRNA概况高度偏向那些对细胞外核糖核酸酶具有固有抗性的RNA。从生物标志物的角度来看,这些高度抗性的exRNA很有趣,但不能代表释放到细胞外空间的RNA的实际体积。为了了解exRNA动态并捕获稳定和不稳定的RNA,我们开发了一种基于大小排阻色谱(SEC)分馏的RNase抑制剂(RI)处理的细胞条件培养基(RI-SEC-seq)的方法。这种方法使我们能够鉴定和研究细胞外核糖体和tRNA,并提供了可以在不久的将来影响生物标志物发现的细胞外RNAome的动态视图。


图形概要:


所述RI-SEC-SEQ协议的概述:大小排阻层析的级分的测序从nonvesicular胞样品用或不用RNA酶抑制剂(+/- RI)


[背景]细胞外RNA(exRNA )参与细胞间通讯,并且在微创液体活检中有望成为疾病的生物标志物(O'Brien et al。,2020)。为了在人体液中运输,exRNA需要获得一定程度的针对细胞外核糖核酸酶的保护,而细胞外核糖核酸酶在细胞外环境中无处不在。迄今为止,最受研究和最了解的机制是RNA封装在细胞外小泡(EVs)内,并将这些内源性脂质纳米颗粒释放到细胞外空间(Kalluri和LeBleu,2020年)。除了简单地保护exRNA免受降解外,EV还包含一系列与细胞摄取有关的表面蛋白(Hoshino等人,2015),作为在细胞之间转移RNA的媒介。这可以在生理上(Thomou等,2017)或在治疗性生物技术设置下(Kamerkar等,2017; Kojima等,2018)发生。

与电动汽车无关的那些exRNA知之甚少(Li et al。,2018)。但是,早期工作表明,人血浆中的大多数microRNA正在与Argonaute 2形成可溶性核糖核蛋白复合物,并且不会在EV内运输(Arroyo等,2011; Turchinovich等,2011)。后来的工作表明,电动汽车的RNA含量没有以前建议的高(Chevillet等,2014),而且我们已经观察到细胞培养基中的大多数exRNA都保留在超速离心上清液中(Tosar等,2015)。

我们惊讶地发现,我们可以在细胞条件培养基的超速离心上清液中测序的大多数小RNA是衍生自甘氨酸和谷氨酸异受体的5'tRNA一半(Tosar等人,2015)。这些序列在血浆,血清和其他生物流体中也是最丰富的(Srinivasan等人,2019),它们似乎也被转运到电动汽车之外(Dhahbi等人,2013)。我们随后观察到这些序列可以形成二聚体,并且这些二聚体对单链核糖核酸酶具有高度抗性(Tosar等人,2018)。这些观察结果表明,细胞外非囊泡RNA谱易于发生生存偏差(Tosar等人,2021),在这里我们倾向于仅观察到对降解更有抵抗力的RNA。尽管这对于生物标记物的研究可能是理想的,但在理解RNA释放机制方面可能会出现问题。因此,我们开发了一种方法来研究非囊泡的exRNA,无论其胞外稳定性如何。这种称为RI-SEC-seq的方法(用于:RNase抑制剂[RI]处理的细胞条件培养基的尺寸排阻色谱[SEC]分级分离)使我们能够研究RNA从细胞中有效释放的形式。按照该程序,我们发现了细胞外核糖体和tRNA的存在,以及细胞外tRNA衍生片段的细胞外生物发生(Tosar等人,2020)。后者由其他人使用不含RNase I的细胞系独立验证(Nechooshtan等,2020)。

在这里,我们提供了实验步骤,以便读者还可以在低,中和高RNase活性下获得细胞外非囊泡RNAome的快照。这将有助于以全面,动态的方式表征exRNA谱,并鉴定具有生物标志物潜力的其他稳定RNA。

关键字:细胞外RNA, RNA生物标志物, 细胞间通讯, 新一代测序, 外泌体, 液体活检, 不同稳定性, 细胞外的核糖体

材料和试剂
1. T-75弹药枪(Corning,目录号:430641U)     
2. 0.22 µm或0.45 µm膜(Millipore,目录号:GSWP04700)     
3. MCF-7细胞系(ATCC,ATCC ® HTB-22 TM ),存储:液氮在不使用时。冷冻保存:遵循ATCC的建议     
4. 1毫升注射器     
5. 14毫升,敞口薄壁超透明管,14 × 95毫米,50Pk(Beckman Coulter Life Sciences,目录号:344060),存储:室温     
6. Vivaspin 20离心浓缩器,MWCO 10 kDa ,PES膜(Sartorius,目录号:VS2001),存储:室温     
7. 96孔标准测定块,2毫升,不含认证的RNase / DNase,用于收集色谱部分(Costar,Corning Incorporated,目录号:3961),存储:室温     
8.的Superdex ® 200 10/300 GL(GE生命科学,西格玛/ Merck的,目录号:GE17-5175-01,停产产物;备选:的Superdex ® 200增加10/300 GL,目录号:GE28-9909-44),储藏:常温     
9. DMEM,高葡萄糖,GlutaMAX TM Supplement (Gibco,Thermo Scientific,目录号:10569010),存储:4°C     
10.胎牛血清(FBS),合格,原产地:巴西(Gibco,Thermo Scientific,目录号:12657029),储存:在-20°C下等分(无菌) 
11. MEGM TM乳腺上皮细胞生长培养基BulletKit TM (Lonza,目录号:CC-3150),保存:基础MEBM培养基保存在4°C 
注意:MEGM SingleQuots TM补充剂储存在-20°C下。混合后,将完整的MEGM培养基储存在4°C直至到期。我们不添加牛垂体提取物或试剂盒中包含的抗生素。

12. DPBS,无钙,镁无(GIBCO,热科学,目录号:14190144) ,存储:4℃ 。使用这种无菌溶液进行细胞培养 
13. DPBS(10 ×),无钙,镁无(GIBCO,热科学,目录号:14200075)。用超纯水或DEPC(焦碳酸二乙酯)处理过的水制成1 ×溶液,过滤,并在尺寸排阻色谱法中用作流动相 
14.胰蛋白酶-EDTA(0.05%),酚红(Gibco,Thermo Scientific,目录号:25300062),保存:-20°C 
15. RNase抑制剂,鼠类(New England Biolabs,目录号:M0314S),存储:-20°C 
16. TRIzol TM试剂(Invitrogen,Thermo Scientific,目录号:15596018),存储:4°C 
17. TRIzol TM LS试剂(Invitrogen,ThermoScientific ,目录号:10296028),存储:4°C 
18. 2-丙醇,用于分子生物学(Sigma-Aldrich,目录号:I9516-500ML),储存:室温。基于TRIzol方法进行RNA纯化所需的试剂 
19.纯净的乙醇,用于分子生物学,存储:室温。基于TRIzol方法进行RNA纯化所需的试剂 
20.糖原,RNA级(Thermo Scientific,目录号:R0551),储存:-20°C 。基于TRIzol方法进行RNA纯化所需的试剂 
21. UltraPure TM不含DNase / RNase的蒸馏水(Invitrogen,ThermoScientific ,目录号:10977035),存储:室温,无菌 
注意:我们不要将这种水用于大量的缓冲液中,例如用于体积排阻色谱的缓冲液。对于这些目的,我们使用军用升的iQ W¯¯亚特我们验证为无RNase的房子。可以使用经DEPC处理的高压灭菌水。在这种情况下,添加DEPC(焦碳酸二乙酯,Sigma,目录号:D5758)至终浓度为0.1%v / v,在室温下孵育过夜,然后高压灭菌以灭活DEPC。

22. 10 × Tris-Borate-EDTA电泳缓冲液(ThermoScientific ,目录号:B52),储存:室温 
23.丙烯酰胺/双丙烯酰胺30%溶液,生物试剂,适用于电泳,29:1(Sigma-Aldrich,目录号:A3574),储存:4°C 。危险(阅读MSDS) 
24. SYBR TM Gold核酸凝胶染料(10,000 × DMSO浓缩液)(Invitrogen,ThermoScientific ,目录号:S11494),存储:-20°C 
25. RNA加载染料,2 × (New England Biolabs,目录号:B0363S),存储:-20°C 
26. Qubit TM RNA HS检测试剂盒(Invitrogen,ThermoScientific ,目录号:Q32852),存储:4°C               
27. NEBNext ®复用小RNA库准备集的Illumina公司® (新英格兰生物实验室,目录号:E7300S) ,存储: - 20℃ 
28. MiSeq试剂盒v2(300个循环)(Ilumina ,目录号:MS-102-2002),存储:-20°C (方框1)和4°C (方框2) 

设备


Novex TM TBE-尿素凝胶(ThermoFisher ,目录号:EC6875BOX)
XCell SureLock微型电池(ThermoFisher ,目录号:EI0001)
ÄKTA纯FPLC系统(Cytiva ,前GE Life Sciences ),具有多个波长检测器(至少能够同时在280和260 nm进行检测)和自动馏分收集器,可容纳96孔测定模块(Costar,2 mL,RNase-免费,货号:3961)
Qubit TM荧光计2.0(Thermo Scientific,目录号:Q32866,停产的产品。由目录号Q33216代替)
MiSeq下一代音序器(Illumina)

软件


注:一个用于测序数据分析软件LL使用银河欧洲Web界面(运行https://usegalaxy.eu)。

fastx_clipper(fastx_toolkit)
Fastqc
RNA之星
BAM过滤器
bamCoverage
featureCounts

程序


注意:即使没有特别说明,在整个实验过程中也应将RNase污染的可能性降到最低。尽可能使用一次性不含RNase的塑料和经认证的不含RNase的溶液。即使在处理非危险试剂时,也应在所有步骤中都戴手套,并在被RNase污染的材料上被RNase污染的表面上工作。


细胞培养
注意:要成功完成此程序,必须在无血清培养基(SFM)中培养细胞,以避免存在血清RNase。在该协议中,我们将使用乳腺上皮生长培养基(MEGM,Lonza)和MCF-7细胞,但是可以将其适应于其他细胞系。但是,为每个细胞系找到合适的SFM并非易事。血清剥夺可引起细胞应激,这会影响细胞内和细胞外RNA谱。如Tosar等人所述。(2020),我们通过蛋白质印迹法检测eIF2亚基α(Ser 51)磷酸化水平,并通过非放射性Northern印迹法监测应激诱导的tRNA衍生片段的水平(Yamasaki et al。,2009)。对于某些细胞类型,可以使用商业SFM制剂。我们已经观察到MCF-7细胞可以在MEGM中短时间生长,MEGM是ATCC推荐用于其他乳腺上皮细胞系(例如MCF-10A)的生长培养基。但是,我们不添加在MEGM试剂盒中获得的牛垂体提取物以将其保留为化学成分确定的培养基。除非需要,否则我们也不会在细胞培养物中添加抗生素。从理论上讲,该协议可以稍加修改即可应用于悬浮细胞,3D培养和类器官,尽管我们尚未尝试过这些应用。

种子近轴。ø NE百万个细胞在每两个T-75烧瓶中的。生长细胞达60 -在完全培养基中70%融合(例如,DMEM + 10%FBS)中在37 ℃下和5%CO 2 。融合应根据每种细胞系的生长速率进行调整。目标是48小时后达到80-90%的汇合度。
洗涤细胞简要3 - 5毫升预热的DMEM或PBS。
加入10毫升预热的MEGM。生长细胞24小时。
再次用预热的MEGM清洗细胞。向其中一个烧瓶中加入10毫升预热的MEGM(以下称对照)。向第二个烧瓶中加入10 ml预热的MEGM + 80U(2 µl )鼠RNase抑制剂(此后:经RI处理),再孵育24小时。
从两个烧瓶中吸出细胞条件培养基,并在15 ml无核酸酶的Falcon锥形管中排出。向RI处理的烧瓶中添加1 µl鼠类RNase抑制剂(仅)。在室温下以800 × g的速度离心细胞5分钟。
虽然离心细胞条件培养基,移动包含细胞单层到通风橱和裂解细胞的空烧瓶2 ml的的Trizol试剂。轻轻倾斜烧瓶以溶解所有细胞,在室温下孵育5分钟,然后移开盖子,将烧瓶储存在-20°C。以后将用于获取细胞内RNA谱。
将来自步骤A 5的上清液放入新的15 ml无核酸酶的Falcon锥形管中,并在4°C下以2,000 × g离心20分钟。我们通常将这次旋转的上清液(保存在新管中)保存在-20 °C供以后使用。

超速离心制备细胞外非囊性样品
注意:超速离心需要在原位进行专门的培训。本协议不能替代此培训。超速离心机使用不当会引起样品丢失,对昂贵设备的永久损坏,并使实验室人员的安全受到威胁。
通过将试管放入室温下装有水的烧杯中,将其从步骤A7解冻。交换几次水,因为水会迅速冷却。
在装有RI处理样品的试管中加入1 µl鼠RNA酶抑制剂。由于重组RNase抑制剂含有许多半胱氨酸并且易于氧化,因此我们通常添加这种增强剂以确保RNase活性不足。
在4°C下以2,000 × g的转速再次旋转样品20分钟,以去除可能在冷冻/融化过程中形成的沉淀物。
将样品放在两个相同的超透明超速离心管中。如果需要,调整样品的用冰冷的不含核酸酶的PBS的体积,使得所述管均达到满负荷减2 -从所述管的顶部为3mm。管路中所有相对的管子必须用相同密度的液体填充到相同的高度。
将试管放在水桶中。我们还检查包含填充的管那水桶显示重量等于或小于2毫克如果它们的差被放置在转子的相对的位置。将铲斗放在已经冷却的转子中(存放在冷藏室中过夜)。将转子放入超速离心机中,设置所有相关参数并开始真空。
注:˚F orgetting冷却转子在这个协议的发展隔夜发挥了重要作用。一位科学记者撰写了有关该情节的故事,提供了一些历史背景(Khamsi ,2020年)。

在4°C下以100,000 × g的速度运行2.5小时,最大加速度和断裂。  
将步骤B 6中的上清液转移至新管中。将试管放在冰上。
使用Vivaspin 20单位(分子量截断值为10 kDa)在4 °C下通过超滤浓缩至500 µl 。在上样之前,请使用不含核酸酶的PBS或水运行超滤装置,以清洁膜。将浓缩液转移至新的无核酸酶的1.5 ml管中。

体积排阻色谱
注意:此过程需要进行两次,使用RI处理的样品和对照样品。使用FPLC系统还需要在现场进行特定的培训,因为将使用高压。避免在所有阶段将空气引入色谱柱,并避免注入含有灰尘或其他微粒的样品或溶剂。

使用真空过滤装置,通过0.22 µm或0.45 µm膜过滤500 ml超纯水和500 ml 1 × PBS(在超纯水中制备)。用电磁搅拌器在减压下对溶液脱气。
在FPLC系统中将警报设置为低于或等于色谱柱允许的最大压力(在这种情况下为1.5 MPa)。将Superdex 200 10/300色谱柱连接到FPLC系统。通过至少1.5体积的过滤和脱气水,从色谱柱上除去乙醇。用1 × PBS平衡色谱柱。
将500 µl定量环连接至系统。用水清洗回路,然后用过滤的PBS清洗。避免引入气泡。
将样品以10,000 × g的转速(从步骤B8开始)旋转4分钟10分钟 ℃。将上清液转移到新管中。
用1M升注射器,注射从样品步骤Ç 4(5在00微升)的列。
通过同时监测280 nm和260 nm的吸光度,以1 ml / min的流速运行。
的0.25收集馏分毫升使用新的,无RNase 96 -孔平板中。开始洗脱至少2 ml,然后洗脱与空隙体积相对应的馏分。
在两个样品中鉴定出的馏分均为ABS 260 > ABS 280 。与蛋白质相比,核酸在260 nm处吸收的紫外线比在280 nm处吸收的紫外线更多。因此,这些级分对应于核酸或核糖核蛋白复合物的洗脱。至少在MCF-7细胞中,对照样品应在Ve = 15 ml处显示一个260 nm的主峰(我们称其为“ P1”:调整后的Ve '= 7.5 ml),在Ve = 16.5 ml处显示一个较低的峰(即我们称为“ P2”:调整后的Ve '= 9.0 ml)。相反,在RI处理过的样品中不应看到P2峰,并且在空隙体积中应出现一个新峰(我们称其为“ P0”:Ve = 7.5 ml;调整后的Ve '= 0 ml)(图1 )。



图1 。RI处理(上图)和对照(下图)样品的代表性色谱图。260 nm和280 nm处的吸光度分别以红色和蓝色显示。280/260> 1的峰对应于蛋白质的洗脱,而280/260 <1的峰对应于核酸和/或核糖核蛋白复合物的洗脱。V e :洗脱体积。改编自Tosar等。(2020年)。


从两个样品中将对应于峰P0,P1和P2的馏分转移至新的1.5 ml无RNase试管中。即使没有明显的峰(例如,对照中的P0或经RI处理的P2),也要确保成对的馏分对应于相同的洗脱体积。
在通风橱中,加入750微升的Trizol试剂LS到每个收集的级分(250微升)。按照制造商的说明进行RNA提取。

RNA分析和下一代测序
将RNA沉淀重悬于9 µl无RNA酶的水中。使用每个样品1微升和Qubit荧光计(按照供应商的说明)检查RNA浓度。如果使用生物分析仪检查RNA完整性,请记住这些不是细胞内样品,因此RNA完整性编号(RIN)的概念在此不适用。然而,经RI处理的样品中的P0峰应包含完整的核糖体RNA。我们不使用Bioanalyzer,而是通过使用0.5 × Tris-硼酸盐-EDTA缓冲液。拌1微升各样品的1微升2的×适于变性polyacrylami样染料德凝胶电泳(图2 )。预制凝胶可以被用作时间效益的替代定制凝胶(例如,的Novex TBE尿素,赛默飞,目录号:EC6875BOX;在所述运行XCELL型Surelock微小区,赛默飞,目录号:EI0001)。



图2.在变性聚丙烯酰胺凝胶中使用(+)或不使用(-)RI处理(左凝胶)或P0峰(右凝胶)的P1峰的分析。细胞:从MCF-7细胞提取的细胞内RNA。显示了对应于tRNA,5S rRNA,5.8S rRNA,7SL RNA,18S rRNA和28S rRNA的条带。左侧的数字(100、200、300)对应于RNA阶梯的迁移(未显示)。凝胶显示P1峰中的RNA从全长tRNA(+ RI)变为大约30 nt的小RNA (-RI)。相反,P0中的RNA较大,并且明显可见28S,18S和5.8S rRNA大小的条带。改编自Tosar等。(2020年)。


使用剩余的7 µl每个样品,使用小型RNA试剂盒(例如用于Illumina的NebNext小型RNA测序试剂盒)进行测序文库制备。在Illumina平台中执行单端测序。在我们的研究中(Tosar等人,2020年),我们进行了200个循环的测序,以便能够基于凝胶电泳结果检测一些全长RNA(例如5.8S rRNA),而不仅是它们的片段(图2 )。 。但是,这样做有一个主要缺点。由于芯片中的大多数簇都包含较小的插入片段,因此测序时间超过150个周期会引发整个适配器的测序,聚合酶的流失以及测序反应的中止终止,然后再进行多路分离,至少在MiSeq台式测序仪中是如此。我们使用的那个。数据分析仍然可以执行,因为索引将包含在每次读取序列的一部分中。然而,从一个被中止运行中提取数据并不简单,我们建议不进行长于100 - 150次,除非理解正在做什么。

清单数据分析小号


如Tosar等人所述分析数据。(2020年)。简要地说,鉴定并剪切3'衔接子序列,将剪切的读段映射到人类基因组,并计数与microRNA,tRNA,rRNA,snRNA,snoRNA,YRNA和Vault RNA对应的特征。可以消除低质量的读数,但不要在读数末尾修剪低质量的碱基,因为这样做会产生使解释复杂化的人工3'变体。RI处理样品与对照样品之间的P1峰比较如图3所示。请注意,在RI处理的样品中28S rRNA的内部片段与全长U49 snoRNA之间鉴定出假定的二聚体,而在对照中则完全不存在。这是RI-SEC-seq如何识别由于细胞外稳定性低而在常规exRNA分析中丢失的细胞外非囊泡RNA复合物的例子(与由30-31个核苷酸的甘氨酸5'tRNA半部分形成的高度稳定的二聚体相比) )(Tosar等人,2018和2020)。





图3 。RI-SEC-seq结果概述。一。小RNA测序的大小分布读取映射到RI处理(上图)或对照样品(下图)的P1峰中的不同ncRNA(参见图例)。RPM:每百万映射读取的读取。数字表示5'tRNA来源的读段的长度。乙。相同,但仅显示与rRNA对齐的读段(红色为28S rRNA,橙色为5.8S rRNA,黑色为5S)。在这种情况下,数字表示大多数读取的起始位置。Ç 。预测SNORD49A的表示(U49A;黑色)/ 28S rRNA的(红色)的相互作用,如在所描绘snoRNABase (www-snorna.biotoul.fr)。以下是读取次数最多的序列。它的相对丰度表示为每百万映射读(RPM)的读。还显示了其在“ P1 + RI”数据集中的排名。改编自Tosar等。(2020年)。


笔记


RI-SEC-seq在exRNA分析中纳入了细胞外稳定性差异的概念。但是,它受到所应用的测序方法的限制。例如,我们知道,在RI处理的样品中P1峰主要包含全长tRNA,这在图2中显而易见。但是,在RI处理的样品中P1的测序未显示出与全酶切相对应的大量读数。长度的tRNA。相反,大多数tRNA衍生的读段对应于5'tRNA一半(图3 )。因此,应将RI-SEC-seq与正交技术(例如Northern blot和密度梯度离心法)结合使用,以获得更具有代表性的细胞外RNAome图片。备选地,本文所述的小RNA测序技术可以替代能够对全长tRNA及其片段进行测序的替代方法,例如TIGRT-seq (Qin等人,2016; Shurtleff等人,2017)。
在P1 + RI和P1 – RI之间,tRNA衍生片段的长度有所变化。的33片段- 34 NT被RI处理的样品中观察到,但它们在对照(丢失图3A )。通过Northern印迹观察到类似的模式(Tosar等,2020)。原因是包含全长tRNA(如PI + RI)的样品的主要切割产物是33' - 35 nt的5'tRNA一半(及其同源的3'一半,通常较难测序)。然而,较短的30 5'半部分- 31趋于稳定得多。这可能是由于二聚体的形成(Tosar et al。,2018)或与细胞外RNA结合蛋白的相互作用,具有大小依赖性。因此,5'的半部30 - 31 NT (主要衍生自的tRNA甘氨酸)往往当RNA酶活动更高积累。因此,RI-SEC-seq可以识别单个RNA类(例如,较短和较长的5'tRNA一半之间)以及不同RNA类之间的不同RNA稳定性的差异(图3A )。
P0(经RI处理的样品)中的小RNA测序显示了多种rRNA衍生片段,主要来自28S rRNA。但是,图2清楚地表明,在P0中洗脱的RNA具有高分子量。应该注意的是,当对主要包含全长长的非编码RNA(例如28S和18S rRNA)的样品进行小RNA测序时,将仅检索其片段。这可能会导致误解,认为exRNA在明显没有的情况下主要是片段化的。我们发现对应于全长5.8S rRNA的大量读物进一步证明了这一点,该读物足够短以适合我们的测序窗口。通过将RI-SEQ-seq与质谱,Western印迹和速度梯度相结合,我们发现P0峰包含由rRNA和核糖体蛋白组成的核糖核蛋白复合物。因此,RI-SEQ-seq方法的发展是导致发现细胞外核糖体的关键步骤(Khamsi,2020; Tosar等,2020)。
该协议的应用扩展到任何情况下,捕获捕获到细胞外空间的整套RNA而不是仅最稳定的RNA(即,甘氨酸或谷氨酸的5'tRNA一半和与EV相关的RNA)都很重要。例如,测试细胞外空间如何在感染细胞和非感染细胞之间,或应激细胞和非应激细胞之间反映细胞内基因表达的变化。此外,RI-SEC-seq是分析细胞外RNA加工机制和exRNA生物发生的有价值的工具。

菜谱


商业试剂并浓缩缓冲液中使用throught这个协议中,上面所指示的。然而,大多数这些缓冲液通常在分子和细胞生物学实验室中使用,并且可以很容易地由普通固体原料制成。在这种特殊情况下,我们选择购买商业解决方案,以在整个方案中将RNase污染降至最低。


致谢


接收到该工作从以下来源资金:AGENCIA国立Investigación ë INNOVACIÓN (ANII,乌拉圭)[FCE_3_2018_1_148745]; Comisión扇形德Investigación Científica (CSIC- UdelaR ,乌拉圭)健康,美国的[MIA_PAS2019_2_99]和国家机构[UG3CA241694,由美国国立卫生研究院共同基金的支持下,通过NIH院长的战略协调/办事处的办公室。JPT和AC是来自PEDECIBA(乌拉圭),国家研究人员系统(ANII)和UdelaR的全心投入计划的研究人员。

该协议的基本原理首先在Tosar等人中提出。(2018),但RI-SEC-seq在Tosar等人中正式开发。(2020年)。该协议中显示的所有数字均摘自后者的出版物。


利益争夺


没有声明


参考


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引用:Tosar, J. P., Gámbaro, F., Castellano, M. and Cayota, A. (2021). RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities. Bio-protocol 11(4): e3918. DOI: 10.21769/BioProtoc.3918.
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