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Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds, however measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprised of target-specific probes and sequentially-hybridized amplification reagents; resulting in a theoretical 8000 – 16,000-fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures leaving the assayed cells intact, to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating the low abundance of mRNA with many biological measurements at the single-cell level.
The genome has become an increasingly accessible repository of information for the academic and clinical study of disease etiology, and its detection and diagnosis. As part of the modern bioinformatics revolution, a vast amount of knowledge has been garnered from transcriptomic technologies such as Microarray, Next Generation Sequencing and Whole Transcriptome Shotgun Sequencing; enabling high resolution and insight into the genome. While these advanced technologies can yield comprehensive gene expression data, their most significant shortcoming lies in the fact that unless a pre-0sorted population of cells is obtained in advance (i.e. via cell sorting), the transcriptional analysis of bulk samples will be obscured with large amounts of data generated from irrelevant cell populations.
Integrating the measurement of genomic expression via the Branched DNA assay with a discriminative technology such as flow cytometry represents an elegant solution to the problem of sample heterogeneity, as multiparametric flow cytometry permits the simultaneous evaluation of mRNA and protein expression at the single-cell level (Buckingham and Flaws, 2007; Wang et al., 2012). The advent of Branched DNA technology complements flow cytometry by allowing for many determinations that were previously unachievable. Of significance, is the ability of Branched DNA technology to label cell targets for which antibody reagents do not exist; whether because the determinants are novel, represent alternative splice variants, or would otherwise require complicated and sometimes inconsistent antigen-retrieval techniques.
In its simplest form, the combination of Branched DNA technology with flow cytometry can be employed for qualitative and semi-quantitative determinations such as the characterization of cellular targets of viral infection, with a concomitant quantification of their viral load. This technology is also ideal for correlating mRNA and protein levels, for studying their expression kinetics, their respective half-lives, and also for verifying the effectiveness of mRNA silencing or regulatory interventions. The ability to measure gene expression by flow cytometry creates many opportunities for efficient investigations of nucleic acid expression characteristics in heterogeneous populations.
Capitalizing on the simultaneous and correlated detection capabilities of multiparametric flow cytometry allows for the determination of how much mRNA is being transcribed, and which specific cells are expressing the interrogated mRNA sequences. Data of this nature can be compared individually or in combination with the aforementioned measurements which can already be performed by flow cytometry. In this regard, the expression profiles of multiple mRNA species in distinct and phenotypically-defined cellular subsets can be correlated with metrics such as cell cycle progression, apoptosis, protein phosphorylation state, signaling kinetics, downstream protein expression; and the cellular response to pharmacologic agents, therapeutic interventions, stimulation, suppression, or other environmental conditions. Branched DNA technology represents an opportunity to explore data correlating the potentially small quantities of mRNA with many biological measurements.
Clinically, this technique might be applied to the detection of chimerism in a recipient host from an unsorted sample, or in quantifying viral load in infected cells, or in calculating the fraction of a cell sample that expresses tumor-specific genes, or exhibits other abnormalities that result in aberrant or elevated mRNA expression (Garcia-Morales et al., 1997). Theoretically, no barrier exists to the identification of target mRNAs, provided that their sequences are known.
An important advantage to the use of flow cytometry for the measurement of mRNA species is the fact that many individual cells can be readily interrogated for the expression of a transcript. Modern clinical flow cytometry has become increasingly driven towards improved detection of minimal residual disease, with current detection sensitivities of 0.01% and proposed methodological advances approaching 0.001%; which rivals the assay sensitivity of PCR-based detection (Arroz et al., 2015; Neale et al., 2004; Weng et al., 2013). Accordingly, the flow cytometry-based Branched DNA technique represents a unique approach to the detection of rare events having clinical significance.
The following protocols provide detailed instruction for performing the Branched DNA procedure using human peripheral blood mononuclear cells (PBMCs). Therein, the BASIC PROTOCOL illustrates the requisite steps to quantity mRNA within cells by flow cytometry, using the measurement of CD8 mRNA in PBMCs as an example. To elucidate which cells are producing mRNA, a proof-of-principle experiment is demonstrated in ALTERNATE PROTOCOL 1, which combines cell surface immunophenotyping with mRNA measurement by flow cytometry. ALTERNATE PROTOCOL 2 extends this assay to include the additional measurement of intracellular proteins, and an example is provided which details the correlated analysis of T-bet and EOMES transcription factors with CD8 mRNA in phenotypically defined lymphocyte subsets.
In situ hybridization is a widely used technique that employs complementary DNA or RNA probes to detect specific nucleic acid sequences within cells. Historically, two approaches have been utilized to improve the sensitivity of mRNA in situ detection; they are (1) the amplification of mRNA sequences prior to their hybridization to reporter molecules (i.e. RT-PCR) or (2) the amplification of signal using secondary detection reagents after target sequences have been hybridized to a reporter probe (i.e. in situ hybridization). Unfortunately, amplification bias and also the amplification of background noise have been impediments to the precise and accurate measurement in both methodologies (Wang et al., 2012). Modern Branched DNA technology provides a unique approach to mRNA detection by greatly amplifying the reporter signal instead of the mRNA transcript. In addition to improving assay sensitivity, this novel strategy has been developed to overcome non-specific binding of detection probes.
In brief, cells are fixed and permeabilized using a ‘Fixation Buffer’ to preserve mRNA sequences of either human or mouse cells in suspension. During the first hybridization step, a pair of mRNA-specific oligonucleotide ‘Target Probes’ with each contain approximately 20 complementary DNA bases is annealed to the targeted mRNA sequence (Figure 1). These Target Probes are designed to hybridize adjacent to each other on the mRNA sequence, in order to increase the specificity of the detection system, as both probes must be in proximity to each other to permit signal amplification to proceed. An additional layer of specificity is conferred when ‘Pre-Amplifier’ molecules are added, as these molecules will only form a stable complex in the presence of both hybridized Target Probes. The detection system is further augmented using ‘Amplifier’ molecules, which hybridize to the branched DNA structure through complimentary pairing to multiple sites on the ‘Pre-Amplifier’ molecule. Finally, ‘Label Probe’ oligonucleotides conjugated to Alexa Fluor® 488, Alexa Fluor® 647 or Alexa Fluor® 750 are hybridized to multiple target sites on ‘Amplifier’ molecules. This comprises one fully-assembled branched DNA structure. To further amplify the signal, the complete Branched DNA complex is composed of approximately 20 such structures; each harboring specificity to a different sequence on the mRNA. The actual number of these structures will depend on the length of the mRNA species being interrogated. Accordingly, the complete Branched DNA complex will theoretically result in an 8,000 to 16,000-fold amplification of signal from a single mRNA transcript. Labeled samples are then acquired by conventional flow cytometry.
Fresh, EDTA or sodium heparin anti-coagulated human blood, bone marrow, or leukoreduction filter retentate specimens may be used in this protocol. Samples should be stored at room temperature for less than 48 hours.
Although this procedure has been optimized for use with human lymphocytes and monocytes, the technique has also been implemented with mouse splenocytes, thymocytes, bone marrow and secondary lymphoid tissues. Additionally, this protocol has been successfully executed with certain adherent (HeLa, PC9) and suspension (U937, Jurkat) human cell lines. Fresh, stimulated, and cryopreserved cells can also be employed as suitable specimens (Hanley et al., 2013; http://www.ebioscience.com/resources/faq/flowrna-faq.htm; Porichis et al., 2014; Van Hoof et al., 2014).
Unless otherwise specifically indicated, all reagents are part of a Prime FlowRNA kit that is marketed by Affymetrix (Catalog # 88-18009-210). The use and mention of this product in the following protocols does not represent an exclusive endorsement of this brand. Rather, the aforementioned kit is the only commercially-available product for this application at the time of this protocol’s writing. For the purposes of maintaining neutrality, subsequent reference to the kit will generically be termed ‘Branched DNA’.
It is extremely critical to establish and maintain a temperature of 40 ± 1 °C in the hybridization oven, in order to obtain successful annealing of nucleic acid components of the Branched DNA complex. A milled aluminum block is employed to facilitate the conduction of heat to the microcentrifuge reaction tube, which also assures rapid equilibration of the reaction mixture to the necessary temperature. If desired, a small amount of water can be added to the well of the milled aluminum block to enhance heat transfer from the block to the reaction tube. It is important to utilize equipment that can re-establish proper temperature specifications within 5 minutes after opening the chamber door to accommodate the placement of samples. The use of a NIST-traceable temperature probe allows for the accurate monitoring and calibration of temperature during the re-equilibration of the chamber.
The specimen requirements for ALTERNATE PROTOCOL 1 are the same as those for the BASIC PROTOCOL
The Branched DNA methodology allows for measuring a correlation between the product of nucleic acid transcription and translation on unsorted samples. ALTERNATE PROTOCOL 1 describes the simultaneous measurement of mRNA transcripts and cell surface antigens via detection by flow cytometry. Correlated measurement of CD8 protein and message on lymphocytes are presented as an example. Before starting an experiment, care should be taken to ensure that the phenotyping reagents and antibodies are compatible with the Branched DNA procedure. Refer to SUPPORT PROTOCOL 2 for more details. As an extension of ALTERNATE PROTOCOL 1, a kinetic study of CD8 mRNA and CD8 protein expression was evaluated in stimulated PBMC subsets (see SUPPORT PROTOCOL 3). In this experiment, it was observed that high expression of CD8 mRNA correlated with high CD8 protein expression in lymphocyte subsets.
The specimen requirements for ALTERNATE PROTOCOL 1 are the same as those for BASIC PROTOCOL
Refer to BASIC PROTOCOL
Refer to the BASIC PROTOCOL, and ensure that the cytometer is capable of measuring fluorescence emission from cells labeled with these additional fluorochrome-conjugated mAbs
The temperature of in situ flow cytometry techniques has previously been reported to adversely contribute to increased autofluorescence in acquired cell populations (Mutty et al., 1999). Environmental conditions such as the fixation reagents that are associated with the protocol have also been implicated in the attenuation of measured signal from fluorochrome-conjugated antibody reagents. SUPPORT PROTOCOL 2 describes an experiment that should be performed to evaluate the effect of the Branched DNA process on the levels of autofluorescence in measured detection channels, as well as the fluorescence intensities of all utilized mAbs.
The specimen requirements for SUPPORT PROTOCOL 2 are the same as those for ALTERNATE PROTOCOL 1
Refer to ALTERNATE PROTOCOL 1
Refer to Table 3 for a comprehensive list of fluorochrome-conjugated mAbs that were tested
Refer to ALTERNATE PROTOCOL 1
The results of these example experiments (Figures 4 – 6) demonstrated that the autofluorescence of interrogated cell samples increased in certain detection channels; and that particular clones, antigens or fluorochromes are more susceptible to the Branched DNA procedure; resulting in an overall decrease in the ability to resolve positive from negative populations (i.e. a decreased Stain Index). Investigators should perform related evaluations to ascertain if their reagents experience similar effects.
An advantage of Branched DNA technology is that it permits the simultaneous measurement of mRNA and protein expression dynamics, which can change considerably following cell stimulation. Prior work for example, has elegantly demonstrated the kinetics of IFNγ upregulation in CD4+ T cells following stimulation with PMA and ionomycin (Van Hoof et al., 2014). T cells can also be activated with mitogens, calcium ionophores and presented antigens. A convenient method of stimulation is to cross-link the T cell receptor using anti-CD3 and anti-CD28 mAbs (Tario et al., 2011). In this SUPPORT PROTOCOL, the kinetics of CD8 mRNA expression in stimulated PBMCs is correlated with CD8 protein expression on CD8 T cells, NK cells, and monocytes.
As shown in Figure 7, CD8 mRNA was generally observed to be expressed to varying degrees in lymphocytes, NK cells, and monocytes. As expected for lymphocytes (CD14− CD56−), the expression of CD8 mRNA was observed to positively correlate with the expression of CD8 protein at all time points in the study. A similar observation was noted for NK cells (CD14− CD56+). CD8 protein was also detected in monocytes (CD14+ CD56−), and this finding is consistent with another published report (Gibbings et al., 2007). The presence of CD8 mRNA in monocytes corroborated the measurement of CD8 protein.
To characterize the cellular localization pattern of CD8 mRNA in all 3 leukocyte subsets, labeled samples were acquired using ImageStream cytometry. The intracellular staining of CD8 mRNA was represented by the punctate and well-segregated red fluorescence signal shown in Figure 8. A spot count of mRNA signal was performed for each acquired cell and the cumulative fluorescence intensity for these spots was subsequently calculated. A linear regression analysis (R2 = 0.9879) demonstrated that a higher cumulative fluorescence intensity of mRNA could be explained by the proportionate increase in the overall number of detected spots, and was not due to an elevated intensity of a single spot.
The specimen requirements for SUPPORT PROTOCOL 3 are the same as those for ALTERNATE PROTOCOL 1
Refer to ALTERNATE PROTOCOL 1
Refer to ALTERNATE PROTOCOL 1
Transcription factors are proteins that localize in the cytoplasmic and nuclear compartments. They play a major regulatory role by activating or repressing the rate at which genetic information is transcribed from DNA into mRNA, therefore they can be regarded as important modulators controlling the expression profile of cells. Given this important regulatory function, interest in deciphering the expression patterns of transcription factors in immune cells has grown tremendously, and should provide novel opportunities to better understand the immune system.
Traditionally, bulk analytical methods such as western blot have been employed to measure transcription factors, but the inability to resolve data due to leukocyte heterogeneity, and the modest sensitivity and specificity of such methods have hampered the application of these techniques. An example of this is illustrated by the study of Forkhead box P3 (FOXP3) which was initially believed to be expressed by CD4+ T regulatory cells. However, using the discriminating power of multiparametric flow cytometry to elucidate the expression of FOXP3, it became apparent that FOXP3 was not restricted to T regulatory cells, but could also be found in other leukocyte subsets. It was determined that the expression of FOXP3 could instead be the consequence of cellular activation, and was even found to be expressed in many tumors (Liang et al., 2015).
The measurement of transcription factors by flow cytometry has typically required specialized buffer systems. The current version of the Branched DNA assay enables the detection of cell surface proteins and intracellular proteins in conjunction with the measurement of mRNA. ALTERNATE PROTOCOL 2 describes the use of this methodology to detect cell surface expression of CD8 and CD56 with intracellular T-bet and EOMES proteins and also CD8 mRNA. These transcription factors were investigated because studies have shown that T-bet and EOMES play regulatory roles in T cell and NK cell differentiation (Buggert et al., 2014; Knox et al., 2014; McLane et al., 2013).
Results from this experiment are presented in Figure 9 and are consistent with prior observations, demonstrating that the expression levels of CD8 protein and CD8 mRNA were positively correlated with each other (r2 = 0.770). Two distinct populations were observed in the CD8Bright CD56− lymphocyte subpopulation (i.e. CD8+ T cells); one population was EOMES− and T-bet−/dim, while the other population was EOMES+ and T-bet+. The CD8− and CD8dim NK cell populations were heterogeneous for EOMES expression, and exhibit the highest T-bet expression level of all CD56+ NK cells. Only the CD8bright NK cells were positive for EOMES, whilst exhibiting attenuated T-bet expression. The expression of EOMES and T-bet in CD8bright NK cells corresponded with the transcription factor expression profile in the CD8+ T cell population. This observation is consistent with immunologic dogma, as these transcription factors have been reported to play a role in the cytolytic maturation of both effector CD8 T cells and NK cells (Intlekofer et al., 2008).
The specimen requirements for this ALTERNATE PROTOCOL 2 are the same as those for the BASIC PROTOCOL.
Refer to ALTERNATE PROTOCOL 1.
Refer to the BASIC PROTOCOL, and ensure that the instrument is capable of measuring fluorescence emission from cells labeled with these additional fluorochrome-conjugated mAbs
RNA represents one of the cell’s most important messaging networks, and the transcriptome is believed to reflect the condition of a cell’s active functional state at any given point in time (Cox and Mann, 2007; Tyakht et al., 2014). The levels of different mRNA molecules within a cell are dynamic, and are more quickly responsive to external stimuli than downstream protein expression. For example, the detection of IFNγ mRNA in activated lymphocytes has been measured using the Branched DNA assay to precede the expression of protein by 90 minutes (Van Hoof et al., 2014). It is noteworthy that the transcriptome is subjected to post-transcriptional modification and for this reason, complementarity and not necessarily congruence, between transcriptome and proteomic profiles could yield important information related to protein synthesis and regulation pathways; particularly if this data can be measured in a correlated manner (Hanley et al., 2013; Unwin and Whetton, 2006). Studies have shown that both mRNA and protein expression levels can serve as independent prognostic indicators of outcome in both malignant and viral disease states, however the generation of such data through traditional mRNA analysis techniques has hitherto been laborious and time-consuming (Bishop et al., 2012; Rondeau et al., 2015; Ukpo et al., 2011; Walter et al., 2015).
Historically, the measurement of mRNA from biological samples has required several different steps, including tissue homogenization, nucleic acid extraction and purification; followed by Northern blotting or PCR analysis after an additional reverse transcription step. Owing to the fact that large amounts of nucleic acids are required for such analyses, these uniparametric approaches are generally compromised by a high degree of sample heterogeneity from cells of disinterest that invariably contaminate bulk material. Further, processing also disrupts the native tissue architecture, therefore spatial information related to transcript expression patterns is necessarily lost.
To preserve spatial information related to nucleic acid expression, and also to minimize potential sample contamination requires an in situ measurement within individual cells. This approach also minimizes the risk of RNase-mediated sample degradation, which is a bane of bulk nucleic acid detection methodologies (Klemm et al., 2014). The first investigations of this nature were reported in 1969, when Gall and Pardue described the use of RNA probes to target specific DNA sequences through a process termed in situ hybridization (Gall and Pardue, 1969). At this time, the probes were detected with radiolabels, which was a lengthy and labor-intensive procedure (Koopman, 2001). Advancements in radiolabeling techniques eventually permitted the detection of only a few dozen mRNA molecules per cell (Harper et al., 1986).
Subsequently, a number of technological and methodological advancements in nucleic acid detection strategies have propelled the field to its current state. A comprehensive review of these developments is beyond the scope of this discussion, but improvements were generally aimed at increasing assay sensitivity, whilst reducing the measured background of alternatives (i.e. fluorescence) to radioactive detection modalities (Goolsby et al., 2000; Holtke and Kessler, 1990; Wiegant et al., 1991; Yang et al., 1994; Yang et al., 1995).
A flow cytometric approach offers a number of logistical improvements over traditionally-employed methodologies, such as faster sampling rates, and improved detection of rare events; with the ability to statistically characterize acquired populations. Events are also interrogated at the single-cell level, which allows for evaluation of the heterogeneity of the measured cell populations. The first report of this nature employed a modified fixation reagent (dimethylsuberimidate), which allowed for nucleic acids to be probed and measured without nuclear disintegration (Trask et al., 1985). Cells processed under these conditions were insensitive to the elevated temperatures and high salt concentrations required for probe hybridization. Detection of nucleic acid sequences was facilitated by the use of a 3-step amplification system that targeted abundant, repetitive DNA; a strategy that was the hallmark of other successful studies (Borzi et al., 1996; Trask et al., 1985; van Dekken et al., 1990).
The detection of mRNA by flow cytometry was first described with single-color experiments which quickly and predictably evolved into the multiplexed detection of mRNA; where 3 separate message species could be reliably detected, and their expression levels could be correlated in parallel samples that were evaluated for DNA content (Bauman and Bentvelzen, 1988; Bayer and Bauman, 1990; Yu et al., 1992). This represented a tremendous achievement, as fluorescence detection signals from mRNA were historically dim, even in specimens with high copy number viral infections (Gibellini et al., 1997; Li et al., 1994; Yu et al., 1992).
A notable advancement contributing to the success of the multiplexed adaptation was the utilization of directly-conjugated fluorescent probes, a strategy that was effective for highly-expressed message. For transcripts expressed at low levels, the use of several separate directly-conjugated probes that bound different regions of the same target was found to increase detection signal as compared to the use of a secondary detection reagent (Koopman, 2001; Tsukamoto et al., 1991; Yu et al., 1992). Methodologically, this is of particular importance as most mRNA species are present in cells at fewer than 50 copies each (Hanley et al., 2013; Levsky and Singer, 2003; Zhang et al., 1997).
Another important strategic development in measuring DNA and mRNA by flow cytometry is represented by the in situ PCR amplification of targeted nucleic acid sequences (Chen and Fuggle, 1993; Patterson et al., 1993). A landmark report demonstrated that single-copy HIV DNA target could be suitably amplified, and detected in a highly-specific assay, where cells containing the gene could be readily-discriminated from negative events (Patterson et al., 1993). Separately, the study proved that low-copy number mRNA sequences could also be similarly-amplified in situ, and that data from these two measurements could be combined to discriminate populations of productively infected, versus transcriptionally quiescent infected cells. The incorporation of cytometry-based in situ PCR with concurrent immunophenotyping was met with limited success as the high temperatures achieved during thermal cycling impart a deleterious effect on fluorochrome and protein stability (Mutty et al., 1999). A clever solution to this problem was to employ a biotin – streptavidin detection system, which allowed for the initial labeling of cell surface targets using a biotinylated antibody. After the PCR reaction, a fluorescent streptavidin reagent could be safely added; however, this approach limited flow cytometric immunophenotyping to just one antigenic determination (Patterson et al., 1995).
Measuring low-count mRNA transcripts concomitant with multiparametric cell surface epitope detection could alternatively be accomplished at lower temperatures, and without amplification through the use of a pool of fluorescently-labeled mRNA probes that target multiple mRNAs with several probes for each mRNA. In a striking example of the power of this approach, investigators demonstrated that while infected CD4+ T cells and CD14+ monocytes both harbored HIV DNA, only the monocytes were transcriptionally active, even during late stage disease progression (Patterson et al., 1998). These observations identified an unappreciated reservoir of replicating HIV virus, and clarified persistent questions surrounding the role of leukocyte subsets at different stages of infection.
Recently, efforts have refocused on amplification strategies, however the target of this amplification has become the probe detection system, instead of the mRNA sequence, itself. This procedural modification is known as the ‘Branched DNA’ assay, which utilizes a series of amplification steps for achieving high sensitivity and specificity (Collins et al., 1997; Kapke et al., 1997; Player et al., 2001; Wang et al., 2012). This refinement resulted in the profound amplification of specific mRNA signals, while considerably suppressing the non-specific background noise that has otherwise plagued prior methodologies (Porichis et al., 2014). Interestingly, this approach also allowed for the use of far lower nucleic acid annealing temperatures (40°C), than those that are utilized with conventional in situ PCR protocols, thus permitting the simultaneous and correlated measurements of multiple mRNA species and antigens.
The improved sensitivity and specificity of the Branched DNA technique has resurrected interest in the detection, quantification, and visualization of mRNA molecules in single cell suspension by flow and imaging cytometry (Hanley et al., 2013; Porichis et al., 2014). The ability to multiplex the detection of mRNA with pre-existing flow cytometric methodologies represents a major development in the ability to characterize cellular responses to environment, disease and therapeutic intervention.
The implementation of Branched DNA assay for flow cytometry is a relatively new technology that was adapted from microscopy. Certain precautions must be taken to prevent adverse experimental outcomes. This section describes critical parameters that should be optimized individually prior to the execution of a Branched DNA experiment in order to generate quality results that are neither skewed nor distorted due non-specific or weak-to-negative mRNA signal. Refer to Internet Resources Section for a link to a detailed troubleshooting guide. The points below are the most important to consider:
Typical results for the measurement of mRNA, surface proteins, and intracellular proteins when samples have been labeled using the Branched DNA technique are presented in Figures 2, ,3,3, ,7,7, and and9.9. In practice, the results obtained from separate experiments may differ considerably from these examples, depending upon the nature of the experiment that is performed (e.g. the mAbs used, the biological specimens tested, and the experimental conditions employed). For any experiment that employs the Branched DNA labeling technique, it is recommended to include FMO controls as the standard reference for region placement; in order to facilitate the discrimination of positive events from negative events. As demonstrated in the example experiment described in SUPPORT PROTOCOL 2, exposure of labeled samples to the Branched DNA assay may adversely affect the detection of fluorescently-conjugated mAb, by either increasing background levels, decreasing measured fluorescence signals, or both. The results associated with these observations are presented in Figures 4 – 6.
The number of experimental samples included in the investigation will influence the actual length of the experiment. A typical full-length Branched DNA procedure using the BASIC PROTOCOL will require a minimum of 10 hours to complete. Inclusion of the cell surface labeling steps (ALTERNATE PROTOCOL 1) will add an additional 1.5 hours to the procedure. Execution of the intracellular labeling process (ALTERNATE PROTOCOL 2) will add another hour to the assay.
As described above, the Branched DNA assay has several Stopping Points when samples can be stored and processing can subsequently be resumed at a later time. For potential Stopping Points, see Steps 17 and 22 of the BASIC PROTOCOL.
The authors would like acknowledge Susan Reynolds for her contributions to the success of this project. Joseph D. Tario, Jr. is an ISAC Scholar. Paul K. Wallace is partially supported by the Roswell Park Cancer Institute Ovarian SPORE NIH Grant 1P50CA159981-01A1. Flow cytometry was performed at Roswell Park Cancer Institute’s Department of Flow and Image Cytometry, which was established in part by equipment grants from the NIH Shared Instrument Program, and receives support from the Core Grant (5 P30 CA016056-29) from the National Cancer Institute to the Roswell Park Cancer Institute.
Hanley, M.B., Lomas, W., Mittar, D., Maino, V., and Park, E. 2013. Detection of low abundance RNA molecules in individual cells by flow cytometry. PLoS One 8:e57002.
Porichis, F., Hart, M.G., Griesbeck, M., Everett, H.L., Hassan, M., Baxter, A.E., Lindqvist, M., Miller, S.M., Soghoian, D.Z., Kavanagh, D.G., Reynolds, S., Norris, B., Mordecai, S.K., Nguyen, Q., Lai, C., and Kaufmann, D.E. 2014. High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat Commun 5:5641.
Van Hoof, D., Lomas, W., Hanley, M.B., and Park, E. 2014. Simultaneous flow cytometric analysis of IFN-gamma and CD4 mRNA and protein expression kinetics in human peripheral blood mononuclear cells during activation. Cytometry A 85:894–900.
Wang, F., Flanagan, J., Su, N., Wang, L.C., Bui, S., Nielson, A., Wu, X., Vo, H.T., Ma, X.J., and Luo, Y. 2012. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14:22–29.