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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Biotechniques. 2015 Jun 1;58(6):308–317. doi: 10.2144/000114299

RiboTag is a flexible tool for measuring the translational state of targeted cells in heterogeneous cell cultures

Adam J Lesiak 1, Matthew Brodsky 1, John F Neumaier 1,*
PMCID: PMC4467021  NIHMSID: NIHMS696260  PMID: 26054767

Abstract

Primary neuronal cultures are a useful tool for measuring pharmacological- and transgene-regulated gene expression; however, accurate measurements can be confounded by heterogeneous cell-types and inconsistent transfection efficiency. Here we describe our adaptation of a ribosomal capture strategy that was designed to be used in transgenic mice expressing tagged ribosomal subunits (RiboTag) in specific cell types, thereby allowing measurement of translating RNA from desired cell types within complex tissues. Using this strategy we were able to isolate and analyze neuron-specific RNA despite the presence of glia by co-transfecting experimental plasmids with plasmids that selectively express RiboTag in neurons. RiboTag immunoprecipitation was capable of recovering high integrity RNA from small numbers of transfected cells that can then be interrogated by a variety of methods (e.g. RT-qPCR, PCR array, RNAseq) and compared to basal RNA expression of the entire culture. Additionally, we demonstrate how co-transfection of RiboTag with sh-RNA constructs can validate and accurately assess the degree of gene expression knockdown, and how RiboTag can be used to measure receptor-mediated gene regulation with transiently expressed DREADD-receptors. RiboTag co-transfection represents a convenient and powerful tool to isolate RNA from a specific subset of cultured cells with a variety of applications for experiments in vitro.

Keywords: Cell-type specific gene expression, translational profiling, RiboTag, TRAP, primary neuronal cultures

Introduction

Measurement of cell-specific RNA expression within complex tissues has represented a significant challenge and requires specialized cell-sorting equipment (e.g. fluorescence-activated cell sorting)(1–3), single-cell PCR(4), a high transfection efficiency or laser capture microdissection of tissue(5, 6). Although these techniques are adequate for many applications, each has significant practical limitations, such as excessive tissue disruption, low yield, degraded RNA, and potential alterations in RNA expression during sample preparation. Techniques such as RiboTag(7, 8), BacTRAP (Translating Ribosome Affinity Purification)(9–11), thiouracil tagging(12, 13), and INTACT (isolation of nuclei tagged in specific cell types)(14) have led to substantial progress in the ability to isolate cell-specific RNA from homogenized tissue samples. Each of these techniques utilizes the same principle, wherein a transgene expresses a “tagged” molecule in a cell-specific manner, and RNA is immunoprecipitated selectively from the cells in which the “tagged” molecule is expressed.

Each of these techniques has advantages and disadvantages, but only the RiboTag and TRAP methods allow for tissue-specific translational profiling. RiboTag utilizes RPL22 conjugated to a hemaglutanin (HA)-tag while the TRAP method uses RPL10 conjugated to eGFP. In both cases, transgenic mice have been designed that express the RiboTag or TRAP transgene in a specific cellular sub-type; however, the utility of RiboTag and TRAP has yet to be explored in vitro.

Translational profiling using RiboTag represents a particularly useful technique in cultures with mixed and difficult to transfect cell-types, such as primary culture of differentiated cells. For example, primary cultured neurons often require co-culturing with non-neuronal glial cells to maintain viability; therefore, any measurements of pharmacological and transgene-regulated gene expression are potentially confounded by detection of RNA in non-neuronal cells or untransfected neurons. Conventional transfection often results in only a small percentage of the neurons expressing transgenes. Attempts to maximize transfection efficiency may compromise cell viability, and low or variable transfection efficiency can hamper experimental replication and data interpretation. While viral-mediated gene transfer is capable of reaching a high rate of transfection efficiency, it is not practical or cost-effective to generate new viral constructs if many transgenes will be tested(4, 15). Additionally, viral mediated gene delivery can be limited by the vector’s payload capacity, which can limit the size and number of transgenes delivered to the target cells(15, 16).

In this report we describe our experience isolating and analyzing translating RNA selectively from transfected cells using primary neuronal cultures as a model system. By co-transfecting RiboTag-expressing plasmids with experimental plasmids, we can improve the sensitivity for detecting changes in gene expression in transfected cells and specific cellular sub-types in vitro. Furthermore, by placing RiboTag under the control of a neuronal specific promoter (Synapsin1)(17), we demonstrate a novel use for RiboTag to monitor neuron-specific, transgene-manipulated, and pharmacologically-induced RNA translation in primary neuronal cultures. Finally, we identify and discuss some of the methodological differences between traditional gene expression analysis and translational profiling, as well as potential pitfalls regarding data analysis and interpretation. We have found that co-transfection with RiboTag offers a flexible method for recovery of RNA from neurons transfected in primary culture, providing a methodology to facilitate enhanced sensitivity of cell-type specific gene expression analysis in vitro.

Materials and methods

Cell culture

Primary dissociated cortical cultures were generated from postnatal day 0–1 C57/BL6 mice (Jackson Labs, Sacramento, CA). Cortical membranes were removed prior to dissociation using papain (Sigma-Aldrich, St. Louis, MO) and glass-polished pipette trituration. Cell density was quantified before being plated at a density of 7 × 104 cells per cm2 in culture dishes pre-coated with poly-L-lysine (Sigma; molecular weight 300,000). Cultures were maintained in Growth Media (GM) consisting of Neurobasal A (NBA) medium (Life Technologies, Carlsbad, CA) supplemented with B27 and Glutamax (1×, Life Technologies) until and throughout treatment days. From the fourth day in vitro (DIV) until homogenization or fixation, culture media was supplemented with 1μM Ara-C (Sigma). This culturing method was adapted from previously described methods(18), and result in cultures consisting of approximately 70% neurons and 30% glia. Cultures were maintained at 37°C at 5% CO2 from DIV0 until homogenization or fixation.

Plasmids and Reagents

The CMV-promoter driven RiboTag construct in pcDNA3 backbone was generously provided by G. Stanley McKnight at University of Washington(7). The hSyn-RiboTag/mRuby2 construct was generated using Gibson Assembly (NEB, Ipswich, MA) following PCR amplification of hSyn-promoter from pAAV-hSyn-Red (Plasmid #22907, Addgene, Cambridge, MA), 2A-skip sequence (ATNFSLLKQAGDVEENPGP) was generated using overlapping oligonucleotides, and mRuby2 PCR amplified from pcDNA3-AKAR2-CR (Plasmid #40255, Addgene). pcDNA3-mRuby2 was generated using the pcDNA3-AKAR2-CR construct, as was pcDNA3-Clover. sh-CREB and sh-scrambled (non-functional sh-RNA construct)(19, 20) were provided by Gary A. Wayman at Washington State University. rM3Ds (Gs-DREADD) construct was provided by Bryan Roth at UNC Chapel Hill(21). Clozapine-N-Oxide (CNO) was provided by the NIMH Chemical Synthesis Program, and forskolin and DMSO were purchased from ABCAM (Cambridge, England). Both forskolin and CNO solutions were made in DMSO and diluted in growth media before reaching final in-well concentration.

Transfection

Neurons were transfected on DIV7 using Lipofectamine 3000 (Life Technologies). Lipofectamine 3000 was added to warm NBA media (0.02μL/μL) and incubated for 5 minutes before combining with pre-mixed total plasmid DNA (50μL/well for 24-well plate and 250μL/well for 6-well plate). Total plasmid DNA for transfections consisted of 1μg/well (24-well plate) and 2μg/well (6-well plate). The percentage of total plasmid transfected for each plasmid-combination used in the experiments was as follows: 50% hSyn-RiboTag/mRuby2, 50% CMV-RiboTag, 10% rM3Ds (Gs-DREADD), 25% CMV-mRuby2, 50% sh-CREB, and for each condition pCAGGS empty vector was added to the plasmid mix to reach 100% of total transfected plasmid. Lipo3K/NBA/DNA mix was incubated at RT for 20 minutes, while native culture media was collected and stored at 37°C and replaced with GM. Lipo3K/NBA/DNA mix was added to plates (50μL 24-well, 250μL 6-well), and incubated for 35–40 minutes before transfection media was aspirated and replaced with original culture media. Transfection efficiencies were less than 2.5% using this transfection protocol in primary neuronal cultures (data not shown). By mixing DNAs prior to addition of the lipofection reagent, micelles are formed with proportionate representation of each plasmid and transfected cells express these plasmids accordingly(22). All transfected cells visualized microscopically demonstrated the expression of all the expected plasmids (Figure 3 and data not shown).

Figure 3.

Figure 3

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Neuronal specificity of hSyn-Ruby/RiboTag plasmid expression and RiboTag-mediated RNA immunoprecipitation. (a) Primary neuronal/glial co-cultures transfected with CMV-Clover(GFP) and CMV-RiboTag or hSyn-mRuby2-2A-RiboTag on DIV7, then fixed and stained for HA on DIV9. Representative images of neurons expressing CMV-promoter driven RiboTag and Clover, neuron expressing hSyn-promoter driven RiboTag and mRuby2, and a transfected glia cell failing to express hSyn-promoter driven RiboTag and mRuby2. Primary neuronal/glial co-cultures transfected with CMV-RiboTag+CMV-mRuby2, hSyn-Ruby/RiboTag or untransfected. RiboTag-IP and Input fractions were analyzed using qPCR to measure normalized relative starting quantity (NRStQ) of neuronal markers (b) Synapsin1 and (c) Map2, and glia markers (d) Gfap and (e) Fabp7. Data shown as RiboTag-IP/Input of NRStQ RNA ± SEM, n=3–5. ANOVA, F(3,12), (a) F=23.45 p<0.0001, (b) F=8.19 p=0.0031, (c) F=15.5 p=0.0002, (d) F=13.22 p=0.0004. Bonferroni Post-Hoc ** p<0.01, * p<0.05 compared to CMV-RiboTag+CMV-mRuby2 and ###p<0.001, ##p<0.01 compared to hSyn-Ruby/RiboTag.

Immunoprecipitation of Polyribosomes

Immunoprecipitation of polyribosomes was conducted as described(7, 8), with minor modifications for use in tissue culture. On DIV9, cultures were administered indicated drug treatments for 1 hour, then neuronal media was aspirated, and cells were washed with 1× PBS prior to being collected in supplemented homogenization buffer (HB: 500μL per well, 50mM Tris-HCl, 100mM KCl, 12mM MgCl2, 1% NP-40, 1mM DTT, 1× Protease Inhibitor Cocktail (Sigma), RNasin 200 Units/mL (Promega, Madison, WI), cyclohexamide 100ug/mL (Sigma), Heparin 1mg/mL (APP pharmaceuticals, Lake Zurich, IL)). Samples were centrifuged at 4°C at 11.9 × g for 10min, and supernatant was collected, reserving 50μL (10%) as input fraction immediately stored at −80°C. 2.5μL of mouse monoclonal HA-specific antibody (HA.11, ascites fluid; Covance, Princeton, NJ) was added to remaining supernatant and RiboTag-IP fractions were rotated at 4°C for 4 hours. 100μL of protein A/G magnetic beads (Promega) were washed with HB prior to addition to RiboTag-IP fraction, and rotated at 4°C overnight. The next day, RiboTag-IP fractions were placed on DynaMag-2 magnet (Life Technologies) on ice, and the bead pellet was washed three times for 5min with high salt buffer (HSB: 50mM Tris, 300mM KCl, 12mM MgCl2, 1% NP-40, 1mM DTT, and 100ug/mL cyclohexamide). After the final wash, HSB was removed and beads were re-suspended in 400μL of supplemented RLT buffer (10μL beta-mercaptoethanol/10mL of RLT Buffer) from the RNeasy plus micro kit (Qiagen, Hilden, Germany) and vortexed vigorously. RLT buffer containing the immunoprecipitated RNA was removed from magnetic beads prior to RNA purification using RNeasy Kit according to manufacturer’s protocol. Likewise, 350μL of RLT was added to Input fractions prior to RNA isolation using RNeasy Kit. Both Input and RiboTag-IP samples were eluted in 14μL of water. RNA Integrity Numbers were determined using High Sensitivity RNA ScreenTape on a 2200 TapeStation (Agilent, Santa Clara, CA) by the Fred Hutchinson Cancer Research Center Genomics Core Facility, RNA Integrity Number = 8.1 ± 0.5 for RiboTag-IP and Input fractions.

RNA Quantification and Reverse Transcription

RNA concentration was measured using Nanodrop and Quant-iT RiboGreen (Life Technologies) methods. 15–30ng of RNA was reverse transcribed using SuperScript VILO Master Mix (Life Technologies). When RNA concentration of negative control sample was great enough, equivalent quantities of RNA was reverse transcribed. If not, maximal allowable volume of negative control RNA was reverse transcribed. For each experimental series, a cDNA standard curve was generated from surplus input fraction RNA then serially diluted to run a standard curve for each gene target. After cDNA synthesis, cDNA was diluted to final working concentration of 0.1ng/μL “relative RNA” (quantity of RNA added to cDNA synthesis reaction).

qPCR

qPCR experiments were carried out on Viia7 Real Time PCR system (Life Technologies) using SYBR Select Master Mix (Life Technologies). Cycling conditions: 50°C 2 min, 95°C 2 min, 40 cycles of 95°C 15 s and 62°C 45 s, followed by a melt curve. For primer list see Supplemental Table 1. Relative starting quantity was calculated based on CT-values of Standard Curve Samples for each series of experiments. Control reactions with reverse transcriptase or template omitted did not amplify. For all analyses except for direct measurement of housekeeping genes (mRuby2, HPRT, PPIA, GAPDH, and ß-Actin), expression was normalized using the housekeeping genes, HPRT and PPIA. Housekeeping gene quantity was consistent across samples within RiboTag-IP and Input fractions, but RiboTag-IP RNA levels were consistently lower than Input. Therefore normalization factors were generated based on average housekeeping levels for RiboTag-IP fractions and Input fractions independently.

Cell Imaging

Primary neuronal cultures grown on glass coverslips were transfected as in RiboTag-IP experiments, except transfected plasmid totaled 1μg/well of 24-well plate, and cultures were fixed for 20 min in 4%paraformaldehyde/PHEMS (Sigma) buffer on DIV10, then washed with 1×-PBS, permeabilized using 1×-PBS with 0.25%TX-100 (Sigma) for 10 minutes, washed again, then mounted with Prolong Gold with DAPI (Life Technologies). Cells were imaged using a Nikon Inverted Microscope (Tokyo, Japan) 40× objective, at the University of Washington Keck Microscopy Facility.

Results and discussion

Primary neuronal/glial co-cultures were transfected with a plasmid co-expressing RiboTag and mRuby2 protein specifically in neurons (hSyn-mRuby2-2A-RiboTag), or co-transfected with CMV-RiboTag and CMV-mRuby2 which should express RiboTag in all transfected cell-types. For negative control conditions, in which no RNA should be immunoprecipitated, cultured cells were transfected with CMV-mRuby2 alone, left untransfected or the anti-HA antibody was excluded from the immunoprecipitation. Only small amounts of RiboTag-isolated RNA are expected to be isolated from primary neurons due to low transfection rate (<2.5%). Isolated RNA varied considerably in RiboTag-IP fractions (~15–100 ng total RNA/well with negative controls ~5–25 ng) whereas total RNA in the Input fractions was >2 μg/well (Figure 1). Despite only collecting small amounts of RNA from RiboTag-IP fractions, equivalent amounts of RNA from RiboTag-IP and Input fractions were reverse transcribed to generate cDNA, to be analyzed by qPCR. When yield was very low (such as in negative controls), the maximal sample volume was reverse transcribed into cDNA alongside experimental samples.

Figure 1.

Figure 1

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Isolation of translating RNA from RiboTag-containing polyribosomes in neuronal/glial co-cultured primary neuronal cultures. (a) RiboTag expression constructs used for experiments. (b) Cartoon of RiboTag protocol for isolation of RNA under active translation as modified for cell culture. Neuronal cultures were transfected on DIV7 with RiboTag-expressing plasmids ± additional plasmids and harvested on DIV9. 10% of this homogenate was reserved as the “Input” fraction, and HA-specific antibody was added to remaining homogenates (except for the no primary antibody control sample). The remainder of the protocol was performed as previously described7.

To confirm successful immunoprecipitation of cell-specific RNA from RiboTag transfected cells, we measured samples for transgenic expression of mRuby2. RNA for mRuby2 was significantly enriched in the RiboTag-IP fractions of RiboTag-expressing neurons compared to negative controls (Figure 2a), while mRuby2 expression was similar across Input fractions transfected with mRuby2-expressing constructs (Figure 2b). Negative control RiboTag-IP samples contained significantly less mRuby2 RNA, although small amounts of mRuby2 mRNA were still detected in both the CMV-mRuby2 and no primary antibody controls. This residual mRuby2 mRNA in RiboTag-IP samples was likely due to non-specific binding of RNA to magnetic beads or insufficient washing during immunoprecipitation. However, no mRuby2 mRNA was detected in either the RiboTag-IP or Input fractions from the untransfected cells.

Figure 2.

Figure 2

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Recovery of RNA from RiboTag-expressing primary neuronal/glial co-cultures, and validation of housekeeping genes. qPCR analysis demonstrating average relative starting quantity of mRNA ± SEM. (a–b) Transgenic mRNA (mRuby2) (a) RiboTag-IP and (b) Input fraction. (c–d) Endogenous housekeeping gene mRNA (c) RiboTag-IP and (d) Input fraction. n=5–8. ANOVA, mRuby2: F(4, 30) (a) F=20.31 p<0.0001, (b) F=12.46 p=<0.0001. HPRT: F(4,31) (c) F=63.46 p<0.0001, (d) F=1.784 p=0.157. PPIA: F(4,31) (c) F=35.55 p<0.0001, (d) F=1.178 p=0.34. ß-actin: F(4,26) (c) F=14.1 p<0.0001, (d) F=0.9 p=0.48. GAPDH: F(4,26) (c) F=59.72 p<0.0001, (d) F=1.68 p=0.19. Bonferroni post-hoc, *p<0.05, **p<0.001 compared to CMV-RiboTag+CMV-mRuby2 and #p<0.05 compared to hSyn-mRuby2-2A-RiboTag.

Housekeeping genes (HKGs) are essential for normalization of qPCR experiments because they provide a standard of comparison between treatment groups, controlling for variability in RNA concentration measurements, reverse transcription efficiency, tissue collection, and RNA integrity(23). Expression of housekeeping genes (e.g. ß-Actin, GAPDH, HPRT, PPIA) ideally should not change between treatment groups. Although, transcription of common housekeeping genes might remain constant, the rate of translation may vary considerably between tissues, cell types, and treatment groups(24, 25). Therefore, we tested expression of four commonly used housekeeping genes in RiboTag-IP and Input fractions collected from CMV-RiboTag and hSyn-RiboTag expressing neurons. Expression of all tested housekeeping genes remained uniform across Input fractions, but varied considerably between neuron-specific and nonspecific promoters (Figure 2c–d). For every housekeeping gene tested, recovery of RiboTag-IP RNA was significantly greater in the CMV-RiboTag transfected cells than in the hSyn-RiboTag transfected cells, even though there were no differences in translation of housekeeping genes across Input fractions (Figure 2c–d). This difference in the translation of HKGs in neurons compared to combined glia and neurons could reflect greater translation of housekeeping genes in glia, or could represent a decrease of neuron-specific RNA when a smaller proportion of the RiboTag-IP RNA comes from neurons. Either way, it is important to ensure appropriate selection of HKGs for RiboTag experiments, especially when comparing different cell populations. Likewise, normalization using HKGs should be conducted within RiboTag-IP and Input samples separately to avoid disproportionately amplifying RiboTag-IP gene expression. HPRT and PPIA were chosen as normalization genes for the remainder of these experiments because ß-Actin has been shown to be translationally regulated in neuronal tissues in some cases(26) and expression of HPRT and PPIA in brain tissues is more stable compared to GAPDH(27).

Attributing a regulated or inducible change in gene expression to a specific cell-subtype in cultures derived from heterogeneous cell-types represents a significant obstacle using traditional techniques. This is especially problematic in primary neuronal cultures that require co-culturing of neurons with glia to maintain neuronal viability. The potential for neuron-specific gene expression to be obscured by glia RNA is a significant obstacle using traditional techniques. Here we demonstrate the utility of RiboTag to isolate neuron-specific RNA simply by restricting RiboTag expression to neurons using a neuron-specific promoter. Immunostaining for RiboTag using an anti-HA antibody determined that neurons in primary cultures co-transfected with CMV-Clover(GFP) and either CMV-RiboTag or hSyn-RiboTag/mRuby2 are positive for anti-HA RiboTag staining, and confirmed that the hSyn-RiboTag/mRuby2 construct expresses un-fused mRuby2 and RiboTag proteins as they were separated by a ribosomal skip sequence(28, 29) (Figure 3a). However, the glia in cultures co-transfected with CMV-Clover and hSyn-RiboTag/mRuby2 did not express RiboTag. We predicted that RiboTag-IP samples collected from hSyn-RiboTag/mRuby2 transfected cultures would be enriched with neuron-specific RNA (Synapsin1 and Map2) and absent of glia RNA contamination (Gfap and Fabp7). Indeed, when RiboTag was expressed via the CMV-promoter, RiboTag-IP fractions contained both neuron and glia RNA (Fig. 3b–e), but when RiboTag was expressed using the neuron-specific promoter, hSyn, the RiboTag-IP fractions were enriched with neuronal RNA, but contained minimally detectable levels of glial RNA (Figure 3b–e). Negative control samples (untransfected cultures or no anti-HA antibody) lacked enrichment of RiboTag-IP RNA for both neuronal and glial RNA transcripts compared to CMV-RiboTag transfected samples. Compared to hSyn-RiboTag transfected samples, negative control samples lacked enrichment of the neuronal RNA transcripts Synapsin1 and Map2 (Figure 3b–e). These experiments demonstrate how restricting RiboTag expression to a specific cellular sub-type can avoid contamination of off-target RNA by simply changing out the gene promoter on the RiboTag plasmid.

Detecting the effects of transgene modulation of endogenous protein function using dominant-negative or constitutively-activate mutants or gene silencing using si/sh-RNAs are common experimental strategies that can be enhanced using RiboTag. Like measuring cell-type specific gene expression, transgene-regulated changes can be obscured by RNA from off-target cell-types, in this case cells not transfected with the transgenes (i.e. background expression of RNAs of interest in untransfected cells can eclipse the detection of changes in transfected cells). To demonstrate this utility, we co-transfected cultures with hSyn-RiboTag and anti-CREB shRNA or control constructs to measure the extent of knockdown of CREB. CREB RNA was significantly reduced by 50% in the sh-CREB transfected RiboTag-IP fractions relative to control, but not in the Input RNA fraction (from all cells) (Figure 4a–c). Thus, RiboTag allows for detection of transgene-regulated changes in gene expression even when a small fraction of the cells are transfected, thereby avoiding false negative results due to unchanged target RNA in the untransfected cells.

Figure 4.

Figure 4

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Transgene and pharmacological experiments using RiboTag. (a–c) RiboTag increases sensitivity of shRNA-mediated knockdown. DIV7 primary neuronal/glial co-cultures were transfected with hSyn-RiboTag and empty vector, sh-CREB or sh-nonsense plasmid. qPCR analaysis and data shown as average normalized relative starting quantity (NRStQ) + SEM of CREB RNA from (a) RiboTag-IP and (b) Input fractions were measured using qPCR. (c) Ratio of RiboTag-IP/Input NRStQ of CREB RNA. Sample sizes were empty vector and sh-CREB n=7, sh-scrambled n=3. ANOVA, F(2,16), (a) F=10.3 p=0.0013, (b) F=1.65 p=0.227, (c) F=4.12 p=0.0391. Bonferroni post-hoc ***p<0.001 compared to Empty Vector. (d–f) Pharmacological regulation of cFos expression. DIV7 primary neuronal/glial co-cultures were transfected with hSyn-Ribotag and rM3Ds (Gs-DREADD) or empty vector, then on DIV9 cultures were treated with vehicle (Veh), 1μM Forskolin (Fsk), or 10μM CNO for 1h prior to homogenization and polyribosome purification. qPCR analysis of average normalized relative starting quantity (NRStQ) + SEM of cFOS RNA from (d) RiboTag-IP and (e) Input fractions. (f) Ratio of RiboTag-IP/Input NRStQ cFos RNA. Sample sizes were n=5–7/group. ANOVA, F(4,25), (d) F=4.612 p=0.0063, (e) F=8.71 p<0.0001, (f) F=3.955 p=0.0071. Bonferroni post-hoc *p<0.05, **p<0.01 compared to Vehicle and ##p<0.01 compared to CNO and rM3Ds condition.

RiboTag can also be used to measure pharmacologically-induced gene expression exclusively in a subset of neurons within heterogeneous cell cultures. When using tissue cultures with homogenous receptor expression, pharmacologically-induced gene expression and translation can be easily measured using standard RT-qPCR and western blot techniques. However, in mixed cultures with heterogeneous cell types and receptor expression, the specific origin of a pharmacologically-induced effect cannot be easily discerned. To demonstrate the utility of RiboTag in pharmacologically-induced gene expression studies, we first treated hSyn-Ruby-2A-RiboTag transfected neuronal cultures with forskolin, a known activator of adenylyl cyclase-dependent production of cAMP that stimulates cFos expression in the majority of cell types(30). As expected, forskolin-treatment induced an increase in cFos RNA in both the RiboTag-IP and Input fractions (Fig. 4 d–f). We then transfected primary neurons with RiboTag and rM3Ds, a Gs-coupled DREADD (Designer Receptors Exclusively Activated by Designer Drugs(21, 31) to activate cFos expression only in transfected cells upon stimulation with the DREADD-specific agonist clozapine-N-oxide (CNO). Neither CNO-treatment nor co-transfection of the rM3Ds alone induced changes in cFos expression in either the RiboTag-IP or Input fractions; however, CNO-treatment of rM3Ds-transfected neurons stimulated an increase in cFos in the RiboTag-IP fraction without changing cFos expression in the Input fraction RNA (Fig. 4 d–f). cFos levels of negative controls were significantly lower than control RiboTag-IP samples, but did not differ from Input controls (data not shown). This experiment demonstrates one of the many ways that RiboTag can be used to measure cell- and receptor-specific changes in transcription (input) and translation (RiboTag-IP) by pharmacological agents in vitro.

RiboTag has the advantage over technologies that strictly measure gene expression, as it allows for simultaneous analysis of the transcriptional (Input fraction) and translational state (RiboTag-IP fraction) in a single experimental sample. This distinction is important, considering that a two-fold increase in translation results in more protein than a thousand-fold change in transcription without a change in translation. With a high rate of transfection, or in cultures derived from RiboTag-expressing transgenic mice, it would be possible to monitor the time course of pharmacologically-regulated transcription and translation. While in many cases it would be expected that transcription precedes translation, RiboTag could aid in the identification of signaling pathways that specifically regulate translation without changing transcription.

Although RiboTag and TRAP were both designed with in vivo experiments in mind(8, 10, 32–37), they are also well suited for a variety of in vitro applications. These methods provide a variety of examples for how RiboTag immunoprecipitation of translating RNA can be flexibly utilized in vitro with great temporal precision despite diverse cellular phenotypes. RiboTag is especially useful when the experiment requires introduction of novel transgenes into difficult-to-transfect cells, and it is necessary to examine the impact of these manipulations just in the transfected cells. One of the highlights of using RiboTag is that special equipment is not required—not every laboratory has access to specialized equipment for laser capture microdissection, fluorescence activated cell-sorting or animal facilities filled with transgenic mice. RiboTag can be utilized in any lab with cell culturing and immunoprecipitation capabilities, and it can be quickly and easily adapted for many cell types simply by changing the promoter driving RiboTag expression. RiboTag represents a convenient and useful tool to monitor dynamic changes in translation, and yields high integrity RNA, even in complex and difficult to transfect cell types like primary neuronal cultures.

Method Summary.

Using primary co-cultures of neurons and glia as a model system, we demonstrate the utility of using RiboTag-expressing plasmids (HA-tagged ribosomal protein) to isolate and measure neuron-specific RNA translation in response to pharmacological and transgenic manipulation without glial-RNA contamination. This method represents a convenient and flexible tool that can be readily implemented for a variety of different applications and cell types without the requirement of specialized equipment or high transfection efficiency.

Acknowledgments

This work was supported by the National Institute on Drug Abuse (NIDA) DA035577, and the estate of Daniel Davis. Support for A.J.L. came in part from NIDA Training Grant 2T32DA00727821.

Footnotes

Author Contributions

A.J.L and J.F.N. designed the experiments and wrote the manuscript. A.J.L. and M.B. executed experiments, and A.J.L. completed data analysis.

Competing Interests Statement

The authors have no competing financial interests to declare.

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