- Email: [email protected]
Chapter 5 Detection of mRNA Species by Flow Cytometry
CHAPTER 5
Detection of mRNA Species by Flow Cytometry Francis Belloc and Franqoise Durrieu Laboratoire d’Himatologie HBpital Haut-Liveque 33600 Pessac France
I. Introduction 11. Applications 111. Materials
IV. V. VI. VII.
A. Synthesis of Fluorescent Oligo(dT) B. Cell Fixation C. RNase Treatment Staining Procedures A. In Situ Hybridization with FITC-o(dT) B. PRINS Labeling with o(dT) as Primer Critical Aspects of the Procedure and Controls Instruments and FCM Analysis Results and Discussion References
I. Introduction Flow cytometry is now widely used for determination of total or nuclear RNA after staining with nucleic acid binding fluorochromes such as acridine orange (Darzynkiewicz et al., 1980), pyronine Y (Darzynkiewicz et al., 1987), thioflavine (Sage et af., 1983), and thiazole orange (Lee et af., 1986). These methods have been employed successfully to study alterations in cellular RNA content during the cell cycle (Darzynkiewicz et al., 1980; Staiano-Coico et al., 1989; Campan et al., 1992) or to discriminate reticulocytes from mature red METHODS IN CELL BIOLOGY. V O L . 42 Copyright 0 1Y94 by Academic Press. Inc , All rights of rcproducrion In any form reserved
59
60
Francis Belloc and Franqoise Durrieu
blood cells (Sage et al., 1983; Lee et al., 1986). However, these staining methods have been little used to follow the effect of drugs on RNA metabolism as they essentially measure ribosomal and transfer RNA, which together make up 90% of the total RNA and are relatively stable species. It would be a great advantage to be able to analyze the effect of drugs on mRNA as the rapid turnover of these species makes them specially sensitive to drugs affecting RNA metabolism. Usually, mRNA is quantified from cell lysates by affinity separation, which exploits the presence of poly(A)+ sequences at the 3’ end of eukaryotic mature mRNA (Jacobson, 1987). Columns of polymers coupled to poly(U) or oligo(dT) are used for their ability to hybridize with the poly(A)+ sequences of mRNA. The bound RNA are then eluted from the column and quantified by spectrophotometry. Unfortunately this method requires a large number of cells, has a variable preparative yield, and has the drawbacks of batch analysis applied to heterogeneous populations. Various attempts to detect rRNA or mRNA by flow cytometry (FCM) have been described using either biotinylated probes and fluorescent in situ hybridization (FISH) (Bauman and Bentvelzen, 1988; Bauman et al., 1990; Bayer and Bauman, 1990) or primed in situ labeling (PRINS) (Mogensen and Kolvroa, 1991; Bolund et al., 1991). Such methods are derived from microscopic and morphologic methodologies. Analysis of FISH and FCM has been hampered by two main problems. First, although the photomultipliers used in FCM can detect low fluorescence intensities, they measure the entire fluorescence signal for each cell and cannot resolve the pattern of fluorescence within the cell. Target-specific fluorescence is not therefore readily discriminated from background fluorescence. The second problem is the “sponge-like” behavior of fixed permeabilized cells in suspension, which tend to trap macromolecules in their cytoplasm. This considerably increases the nonspecific fluorescence when fluorescent proteins such as avidin or antibodies are used to reveal the probes. In view of these drawbacks, a more specific labeling method is required for flow cytometric analysis of FISH, which takes both the characteristics of the detectors and the properties of the cell suspensions into account. In this chapter we describe two methods for revealing poly(A)+ RNA, which are particularly suited to flow cytometric analysis (Fig. 1). FISH using FITC-coupled olido(dT),, [FITC-o(dT)] as a probe: this small, directly fluorescent oligonucleotide enables one-step staining of the cells, thereby reducing cell handling and damage. Background fluorescence is also decreased as this probe is easily washed out of cells. PRINS using oligo(dT) as a primer and reverse transcriptase as a polymerase: the poly(A)+ RNA is used as a template to incorporate FITC-dUTP into complementary strand. Incorporation of fluorescence into a macromolecule is conditioned by hybridization of the primer with the target and so confers high specificity for the fluorescent label.
5. Detection of mRNA by FCM
A
B
61
0.0
3'
Poly-A
mRNA
5'
-00
3'
Poly-A
mRNA
5'
Fig. 1 Schematic representation of the two procedures described for discriminating poly(A)+ RNA by flow cytometry. (A) The oligo(dT) was chemically coupled to a molecule of FITC and directly used as a probe for FISH. (B) The oligo(dT) was used as a primer for PRINS. Labeling was introduced from fluorescein- 12-dUTP by elongation of the primer using reverse transcriptase in siru.
11. Applications Multiparametric analysis can be employed to detect and quantify mRNA by FCM. The different populations from heterogeneous samples can be identified (on the basis of their scatter properties for example) and mRNA content in a given population can by analyzed. Cell membranes can also be immunolabeled prior to fixation of the cells in order to study alterations in mRNA content in a population expressing a particular antigen. The FCM methods developed in our laboratory were designed principally to study the effect of anti-neoplastic drugs on mRNA metabolism. These drugs usually induce a block in one of the phases of the cell cycle. Comparison of the mRNA content in bulk of a blocked cell population with that of a growing population provides little information, as the mRNA content of growing cells varies considerably during the cell cycle (Belloc et af.,1993a). Moreover, some mRNA content of anthracycline-treated
62
Francis Belloc and Franqoise Durrieu
cells (blocked in G2)with growing control cells (which are mostly G,) is meaningless. However, the mRNA content of individual cells and, for example, gated G,M populations, can be assessed by FCM after DNA and mRNA are doublestained.
111. Materials Oligothymidylate [o(dT),,] was purchased from Pharmacia (Saint Quentin en Yvelines, France); chemical reagents were from Aldrich (Strasbourg, France); and FITC isomer I adsorbed on Celite, poly(A), and poly(U) were from Sigma (St. Quentin Fallavier, France). Pancreatic ribonuclease A (RNase) was purchased from Boehringer-Mannheim (Meylan, France) and propidium iodide from Calbiochem (Meudon, France). Fluorescein- 12-dUTP was from Boehringer-Mannheim, and M-MLV reverse transcriptase and the 5 x RT buffer were from Gibco BRL (Cergy Pontoise-France). The promyelocytic leukemia cell line HL-60 was routinely cultured in suspension in RPMI supplemented with 10% fetal calf serum, glutamine, Hepes, buffer, penicillin, and streptomycin. The cells were maintained in exponential growth by dilution to 2 X 105/mlevery other day. A. Synthesis of Fluorescent Oligo(dT) All the solvents must be anhydrous. The 8% cetyltrimethylammonium bromide solution can be stored at 4"C, and must be warmed before use to facilitate dissolution. The FITC adsorbed on Celite and the dipyridyldisulfide (Aldrithiol) were stored at 4°C. The other reagents were stored desiccated at room temperature. The procedure has been described elsewhere (Godovikova et al., 1986). A total of 200 pg of 5' phosphorylated o(dT)(15 mer) was dissolved in 30 pl of distilled water and precipitated as the cetyltrimethylammonium salt by addition of 3 pl of 8% hexadecyltrimethylammonium bromide (CTAB). The precipitate was pelleted 5 min at 10,OOOg. CTAB (1.5 pl) was added and the suspension was centrifuged again. This stepwise addition of CTAB was repeated until no more precipitate was formed. At this stage the oligonucleotide was in its CTAB salt form, which is soluble in DMSO. The pellet was resuspended in dry methanol and evaporated to dryness in a vacuum drier (Speed Vac). The CTAB salt of o(dT) was dissolved in 200 pI of water-free DMSO containing N-methylimidazole (0.6 M ) , dipyridyldisulfide (0.3 M ) , and triphenylphosphine (0.3 M ) . The mixture was incubated for 15 min at room temperature to activate the terminal phosphate, and ethylenediamine was added (1.2 M final concentration) and incubated for a further 30 min. At this stage, the diamine was coupled to the 5' phosphate. The o(dT) derivative was precipitated by adding 1 ml of
5. Detection of m R N A by FCM
63
acetone containing 3% of LiCIO, and the precipitate was washed twice with acetone by centrifugation. The pellet was dried by evaporating the acetone at 60°C and was then dissolved in 100 p1 of 0.3 M triethylamine in water. The 5 ' terminal amino group was coupled to FITC by adding 2 mg of Celite containing 10% FITC and incubating the mixture overnight with constant agitation. The FITC-5'o(dT) was separated from the Celite by centrifugation and precipitated by adding 0. I volume of 3 M sodium acetate, pH 5, and 2 volumes of ethanol. After 1 hr at -20°C the precipitate was recovered by centrifugation for 15 min at 12,OOOg, washed with 70% ethanol, and dissolved in TBE buffer. The fluorescent oligonucleotide was purified from the uncoupled reaction products by electrophoresis on a 20% acrylamide sequencing gel. The brightly fluorescent band corresponding to the FITC-o(dT),, was excised, the acrylamide gel was crushed, and the FITC-o(dT) was eluted overnight with 0.5 ml of 0.1% Triton X-100, 0.3 M LiCIO, in H,O. The FITC-o(dT) was precipitated by addition of 3 ml of acetone containing 3% LiCIO, ( I hr at -2O"C), washed with acetone, dried, and dissolved in 200 pl of H,O. The concentration can be measured by spectrofluorometry, using dilutions of FITC as standard. B. Cell Fixation
FISH: 4 X lo6 HL-60 cells were washed in cold PBS and resuspended in 1 ml of 1% paraformaldehyde in PBS for 5 min at room temperature. After centrifugation (10 min, 200g, P C ) , the cells were resuspended in I ml of PBS, and 2.3 ml of absolute ethanol was added. After centrifugation for 3 min at IOOOg, the cells were resuspended in I ml of 70% ethanol and conserved at -20°C for several weeks. PRINS: prefixation with PFA was found to significantly reduce elongation of the primer by the reverse transcriptase. The samples for PRINS labeling were the fixed at a concentration of 4 x lo6 cells/ml with 70% ethanol. C. RNase Treatment
In some experiments, 4 x lo5 fixed cells were pelleted for 20 sec at 12,OOOg, resuspended in PBS containing 10 U/ml of RNase A, and incubated for 30 min at 37°C. The cells were then pelleted and conserved in 70% ethanol.
IV. Staining Procedures All the following manipulations were carried out in autoclaved conical microtubes; the centrifugations were for 30 sec at 12,OOOg in a microfuge; all solutions were sterilized by filtration at 0.22 pm and prepared with DEPC-treated distilled water.
64
Francis Belloc and Franqoise Durrieu
A. In Situ Hybridization with FITC-o(dT)
Add 3 p1 of a 10% solution of DEPC in ethanol to 50 pi ( 2 x lo5) of the fixed cell suspension. Incubate for 5 rnin at room temperature to inhibit endogenous RNase. Pellet the cells and wash with 100 pI of PBS containing 0.5% Tween 20 (PBST). Add 100 p15x SSPE (1 x SSPE: NaC1, 180 mM; EDTA, 1 mM; Na, HPO,, 5 mM) and leave to equilibrate for 1 hr at room temperature. Pellet the cells. Resuspend the cells in 10 pl of the hybridization mixture (HM): 0.1% SDS, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% albumin, 0.5 mg/ ml calf thymus DNA, and 0.5 pg/ml FITC-o(dT) in 5x SSPE. The mixture was incubated for 2 hr at 35°C under constant agitation. Add 40 pI of H 2 0 and incubate for 30 min at 35°C. Pellet the cells. Wash the cells in 100 pl of PBST; resuspend on 0.7 ml of PBST containing 1 pglml of propidium iodide and 1 Ulrnl of RNase. After 10 rnin at 20°C, the samples are ready for flow cytometry. B. PRINS Labeling with o(dT) as Primer
Pellet 100 pi of fixed cell suspension (4 X lo5 cells) and resuspend in 10 pl H20. Add 1 p1 of o(dT). Incubate for 10 rnin at 70"C, and cool on ice. Add 4 pI of 5 x RT buffer; 2 pI of 0.1 M DTT; 2pl of a mixture of 1 mM of each dGTP, dATP, and dCTP; 0.7 p1 of 1 mM fluorescein-12-dUTP; and 1 pl (200 U) of reverse transcriptase. Incubate for 60 rnin at 37°C. Add 500 pI of PBST. Pellet the cells. Resuspend the cells in 0.7 ml of PBST containing 1 pg/ml of propidium iodide and 1 U/ml of RNase. After 10 rnin at 20"C, the samples are ready for flow cytometry.
V. Critical Aspects of the Procedure and Controls Avoidance of any contamination with RNase is crucial to the success of any work concerning mRNA. The operator must wear gloves throughout the experiments. All the conical microfuge tubes and pipette tips must be autoclaved and kept separate from the rest of the laboratory supply. All the solutions were prepared using DEPC-treated distilled water. The duration of the hybridization step depends on the accessibility of the RNA to the probe and may vary from one experiment to another. It can be affected by the nature of the cells, the size of the oligonucleotide, and the fixation procedure, but 1 hr will usually be sufficient.
5. Detection of mRNA by FCM
65 Table I Verification of Specificity of Fish for Poly(A) ~
+
RNA
~
Sample
Ratio
%
Positive (0.5 pg/ml FITC-o(dT) Negative (no probe) FITC-o(dT) + poly(U)(3 mg/ml) FITC-o(dT) + poly(A)(3 rng/ml) Fixed cells treated with RNase Cells incubated with 1 u d m l Act-D for 6 hr.
3.7 I I .9
100
1.1
1.5 2. I
0 33 4 19 39
Note. The ratio was calculated as the mean fluorescence channel of the sample divided by the mean fluorescence channel of the negative control.
On counterstaining DNA with propidium iodide (PI), very low concentrations of PI must be used (1 to 3 pg/ml are enough to obtain a “cell-cycle shape” on the histogram) to avoid an excessive contamination of the faint green fluorescence by PI fluorescence. The specificity of the label must be assessed by running controls. For FISH (Table I), the specific labeling is sensitive to pretreatment of the cells with RNase; its intensity was significantly decreased by competitive unlabeled probes [o(dT) or poly(U)] and by competitive target [soluble poly(A)]. There was a significant decrease in labeling after incubating the cells with I ,ug/ml of D-actinomycin for 6 hr, showing the accuracy of the method for detecting the effect of drugs on mRNA metabolism. For PRINS (Table II), the labeling was sensitive to pretreatment with RNase and was dependent on the presence of both primer [o(dT)] and polymerase (reverse transcriptase). Excess TTP reduced the labeling, probably by competing with fluorescent dUTP for the transcriptase. Table I1 Verification of Specificity of Prins Sample
Ratio
%
Complete reaction mix Without reverse transcriptase Without o(dT) + 70 pM TTP Fixed cells treated with RNase Cells incubated with I pg/ml Act-D for 6 hr.
4. I
100 0
I 2 2.4 1.8 2.3
32 38 25 44
Note. The ratio was calculated as the mean fluorescence channel
of the sample divided by the mean fluorescence channel of the negative control.
Francis Belloc and Franqoise Durrieu
66
Incubation of the cells with D-actinomycin induced a decrease similar to that observed with the FISH method. For both methods, incubation of the cells with RNase after the labeling step will not significantly affect the fluorescent signal as the labeling (either hybridization or primer elongation) produces an RNase-resistant RNA/DNA hybrid.
VI. Instruments and FCM Analysis For FCM analysis, we used an ATC 3000 cytometer (ODAM-Brucker; Wissembourg, France) equipped with a 2025 Spectraphysics argon ion laser. The beam was tuned to emit 500 mW at 488 nm. The emission was split into green and red fluorescences by a 600-nm short-pass filter (Melles-Griot, Arnhem, Holland). The mRNA-specific green fluorescence (FITC) was collected through a 530 ? 30 nm band pass filter (Oriel, Paris, France). The DNA-specific red fluorescence (PI) was collected through a 600-nm long-pass filter (Melles-Griot). For each experiment, a sample of RNase-treated control cells was labeled and analyzed and the green signal was corrected for red contamination by electronic compensation until the biparametric FITC/PI histogram assumed a horizontal shape (Fig. 2). All further analyses were carried out at this electronic setting. The mRNA content of cells within specific regions of the cell cycle can be determined by gating the signal. For example, a gate can be set on the DNA histogram to provide the mRNA content of G,M cells before and after treatment with anthracycline. The data are collected and stored in list-mode fashion. and
i
4
0
Fig. 2
C
OM
FCM gated analysis of mRNA content on HL-60 cells. FISH using FITC-o(dT) was performed on control cells (A), on cells digested with RNase (B), and on cells treated with 10 nM idarubicin for 24 hr (C). The cells were counterstained with I pg/ml of PI and analyzed by FCM. The red fluorescence (DNA) is on the horizontal axis and the green fluorescence (mRNA) on the vertical axis. The green fluorescence in B was electronically compensated to obtain a horizontal distribution. The samples in A and C were then analyzed with the same electronic settings. The box encompassing the G2M cells was used to gate the analysis of mRNA content in G2M cells. The mean fluorescence value of control (in A) and Ida-treated cells (in C) was corrected for the value of RNase-treated cells (in B) and could then be compared.
67
5. Detection of mRNA by FCM
the measurements can then be retrieved for cells in a given phase of the cell cycle. For accurate measurement of the effect of a drug on the mRNA content, the mean fluorescence channel of the sample was corrected for the value of the RNase-treated sample (RNase-sensitive fluorescence).
VII. Results and Discussion Particular species of mRNA can be detected and quantified relatively by FISH using FITC-o(dT) as a probe followed by FCM (Table I and Belloc et a / . , 1993a). The effect of drugs on mRNA metabolism can be evaluated by multiparametnc analysis with gating on DNA content (Fig. 3 and Belloc et al., 1993b). It can be seen in Fig. 2A that G2M cells contain more mRNA than G, cells. Moreover, the mRNA in G2M are probably different than those of the G, cells (Campan et al., 1992). By gated acquisition, the mRNA content of G2Mcells from idarubicine-treated cells (Fig. 2C) can be readily compared with the mRNA content of G,M control cells (Fig. 2A). The mRNA content was defined as the RNase-sensitive green fluorescence: the mean green fluorescence channel of the defined cell population minus the mean green fluorescence of the same population in the RNase-treated sample. Using this method, we were
E '0
10
20
30
40
50
Incubation time (hrs)
Fig. 3 Flow cytometric analysis of the effect of drugs on the mRNA content. (A) HL-60 cells were treated with 10 ng/ml of idarubicine (open symbols) or 500 ng/ml of actinomycine D (closed symbols) for different periods, fixed, and hybridized with FITC-o(dT). The RNase-sensitive mRNA content of G2M cells was measured by flow cytometry as described in Fig. 2. The results are expressed as percent of initial mRNA content as a function of the duration of the incubation. (B) HL-60 cells were incubated for 8 hr with increasing concentrations of idarubicine. The RNase sensitive mRNA content of G,M cells was plotted as a function of the drug concentration.
68
Francis Belloc and Franqoise Durrieu
able to detect an accumulation of mRNA in Idarubicine-treated HL-60 cells and a decrease in mRNA in actinomycine-D-treated cells (Fig. 3 and Belloc et al., 1993a,b). The tedious and time consuming synthesis of FITC-o(dT) is the main drawback of this method, although it is relatively inexpensive. Much time could be saved by replacing the chemical coupling method with an enzymatic tailing of the o(dT) using terminal deoxynucleotidyl transferase and commercially available fluorescent deoxynucleotide triphosphates. The main drawback of such an enzymatic procedure would be difficulty in controlling the reaction to obtain probes that are homogeneous in size and fluorescence. Dideoxy fluorescent nucleotides triphosphates can be tried for this purpose. The PRINS labeling method using fluorescein-12dUTP as a label, o(dT) as a primer, and reverse transcriptase as a polymerase represents another approach, which in principle is an improvement on the preceding methods (Belund et al., 1991). Hybridization and fluorescence incorporation are performed in a single step; the primer confers the specificity, while the sensitivity is defined by the enzyme efficiency. In addition, primer molecules retained nonspecifically in the cytoplasm do not give rise to fluorescence incorporation in the absence of template, and several fluorescent molecules can be incorporated for each o(dT) molecule hybridized. Together these advantages should produce a very high signallnoise ratio. So far we have only obtained a signal to noise ratio of the same order of magnitude as that of FITC. Table I1 shows that fluorescence incorporation was RNase sensitive, was influenced by treatment of the cells with D-actinomycin, and was dependent on the presence of both o(dT) and reverse transcriptase, which is indicative of good specificity for poly(A)+ RNA. In practice, the PRINS method was found to be a rapid and sensitive way of detecting poly(A)+ RNA by FCM. However, relative quantification is only accurate if one operates within the linear part of the enzyme kinetics. If this condition is met, the incorporated fluorescence should be proportional to the amount of hybridized o(dT). References Bauman, J. G. J., and Bentvelzen, P. (1988). Cyromerry 9, 517-524. Bauman, J. G. J., Bayer, J. A., and van Dekken, H. (1990). J . Microsc. (Oxford) 157, 73-81. Bayer, J. A., and Bauman, J. G. J. (1990). Cyromerry 11, 132-143. Belloc, F., Lacombe, F., Dumain, P., Mergny, J. L., Lopez, F., Bernard, P.,Reiffers, J., and Boisseau, M. R. (1993a). Cyrometry 14, 339-343. Belloc, F., Lacombe, F., Dumain, P., Lopez, F., Bernard, P., Reiffers, J., and Boisseau, M. R. (1993b). Cytometry, Suppl. 6, 38. Bolund, L., Hindkjaer, J., Junker, S., Koch, J., Kolvraa, S., Mogensen, J., Nygaard, M., and Pedersen, S. (1991). Cyromerry, Suppl. 5,61. Campan, M., Desgranges, C., Gadeau, A. P., Millet, D., and Belloc, F. (1992). J . Cell. Physiol. 150,493-500. Darzynkiewicz, Z., Traganos, P., and Melamed, M. R. (1980). Cyromerry 1,98-108. Darzynkiewicz, Z., Kapuscinski, J., Traganos, F., and Crissman, H. (1987). Cyromerry 8,138-145. Godovikova, T. S . , Zarytova, V. F., and Khalimskaya, L. M. (1986). Bioorg. Khimi. U ,475-481.
5. Detection of mRNA by FCM
69
Jacobson, A. (1987). I n “Methods in Enzymology” (S. L. Berger and A. R . Kimmel, eds.), Vol. 152, pp. 254-261. Academic Press, San Diego. Lee, L. G . , Chen. C. H., and Chiuu, L. A. (1986). Cyromerry 7, 508-517. Morgensen, J . , and Kolvroa, S. (1991). Exp. Cell Res. 196, 92-98. Sage, B. H . , Jr., O’Connell, .I.P., and Mercouno, T. J . (1983). Cyfomefry 4, 222-227. Staiano-Coico, L . , Darzynkiewicz, Z.. and McMahon, C. K . (1989). Cell Tissue Kiner. 22,235-243.
Detection of mRNA Species by Flow Cytometry Francis Belloc and Franqoise Durrieu Laboratoire d’Himatologie HBpital Haut-Liveque 33600 Pessac France
I. Introduction 11. Applications 111. Materials
IV. V. VI. VII.
A. Synthesis of Fluorescent Oligo(dT) B. Cell Fixation C. RNase Treatment Staining Procedures A. In Situ Hybridization with FITC-o(dT) B. PRINS Labeling with o(dT) as Primer Critical Aspects of the Procedure and Controls Instruments and FCM Analysis Results and Discussion References
I. Introduction Flow cytometry is now widely used for determination of total or nuclear RNA after staining with nucleic acid binding fluorochromes such as acridine orange (Darzynkiewicz et al., 1980), pyronine Y (Darzynkiewicz et al., 1987), thioflavine (Sage et af., 1983), and thiazole orange (Lee et af., 1986). These methods have been employed successfully to study alterations in cellular RNA content during the cell cycle (Darzynkiewicz et al., 1980; Staiano-Coico et al., 1989; Campan et al., 1992) or to discriminate reticulocytes from mature red METHODS IN CELL BIOLOGY. V O L . 42 Copyright 0 1Y94 by Academic Press. Inc , All rights of rcproducrion In any form reserved
59
60
Francis Belloc and Franqoise Durrieu
blood cells (Sage et al., 1983; Lee et al., 1986). However, these staining methods have been little used to follow the effect of drugs on RNA metabolism as they essentially measure ribosomal and transfer RNA, which together make up 90% of the total RNA and are relatively stable species. It would be a great advantage to be able to analyze the effect of drugs on mRNA as the rapid turnover of these species makes them specially sensitive to drugs affecting RNA metabolism. Usually, mRNA is quantified from cell lysates by affinity separation, which exploits the presence of poly(A)+ sequences at the 3’ end of eukaryotic mature mRNA (Jacobson, 1987). Columns of polymers coupled to poly(U) or oligo(dT) are used for their ability to hybridize with the poly(A)+ sequences of mRNA. The bound RNA are then eluted from the column and quantified by spectrophotometry. Unfortunately this method requires a large number of cells, has a variable preparative yield, and has the drawbacks of batch analysis applied to heterogeneous populations. Various attempts to detect rRNA or mRNA by flow cytometry (FCM) have been described using either biotinylated probes and fluorescent in situ hybridization (FISH) (Bauman and Bentvelzen, 1988; Bauman et al., 1990; Bayer and Bauman, 1990) or primed in situ labeling (PRINS) (Mogensen and Kolvroa, 1991; Bolund et al., 1991). Such methods are derived from microscopic and morphologic methodologies. Analysis of FISH and FCM has been hampered by two main problems. First, although the photomultipliers used in FCM can detect low fluorescence intensities, they measure the entire fluorescence signal for each cell and cannot resolve the pattern of fluorescence within the cell. Target-specific fluorescence is not therefore readily discriminated from background fluorescence. The second problem is the “sponge-like” behavior of fixed permeabilized cells in suspension, which tend to trap macromolecules in their cytoplasm. This considerably increases the nonspecific fluorescence when fluorescent proteins such as avidin or antibodies are used to reveal the probes. In view of these drawbacks, a more specific labeling method is required for flow cytometric analysis of FISH, which takes both the characteristics of the detectors and the properties of the cell suspensions into account. In this chapter we describe two methods for revealing poly(A)+ RNA, which are particularly suited to flow cytometric analysis (Fig. 1). FISH using FITC-coupled olido(dT),, [FITC-o(dT)] as a probe: this small, directly fluorescent oligonucleotide enables one-step staining of the cells, thereby reducing cell handling and damage. Background fluorescence is also decreased as this probe is easily washed out of cells. PRINS using oligo(dT) as a primer and reverse transcriptase as a polymerase: the poly(A)+ RNA is used as a template to incorporate FITC-dUTP into complementary strand. Incorporation of fluorescence into a macromolecule is conditioned by hybridization of the primer with the target and so confers high specificity for the fluorescent label.
5. Detection of mRNA by FCM
A
B
61
0.0
3'
Poly-A
mRNA
5'
-00
3'
Poly-A
mRNA
5'
Fig. 1 Schematic representation of the two procedures described for discriminating poly(A)+ RNA by flow cytometry. (A) The oligo(dT) was chemically coupled to a molecule of FITC and directly used as a probe for FISH. (B) The oligo(dT) was used as a primer for PRINS. Labeling was introduced from fluorescein- 12-dUTP by elongation of the primer using reverse transcriptase in siru.
11. Applications Multiparametric analysis can be employed to detect and quantify mRNA by FCM. The different populations from heterogeneous samples can be identified (on the basis of their scatter properties for example) and mRNA content in a given population can by analyzed. Cell membranes can also be immunolabeled prior to fixation of the cells in order to study alterations in mRNA content in a population expressing a particular antigen. The FCM methods developed in our laboratory were designed principally to study the effect of anti-neoplastic drugs on mRNA metabolism. These drugs usually induce a block in one of the phases of the cell cycle. Comparison of the mRNA content in bulk of a blocked cell population with that of a growing population provides little information, as the mRNA content of growing cells varies considerably during the cell cycle (Belloc et af.,1993a). Moreover, some mRNA content of anthracycline-treated
62
Francis Belloc and Franqoise Durrieu
cells (blocked in G2)with growing control cells (which are mostly G,) is meaningless. However, the mRNA content of individual cells and, for example, gated G,M populations, can be assessed by FCM after DNA and mRNA are doublestained.
111. Materials Oligothymidylate [o(dT),,] was purchased from Pharmacia (Saint Quentin en Yvelines, France); chemical reagents were from Aldrich (Strasbourg, France); and FITC isomer I adsorbed on Celite, poly(A), and poly(U) were from Sigma (St. Quentin Fallavier, France). Pancreatic ribonuclease A (RNase) was purchased from Boehringer-Mannheim (Meylan, France) and propidium iodide from Calbiochem (Meudon, France). Fluorescein- 12-dUTP was from Boehringer-Mannheim, and M-MLV reverse transcriptase and the 5 x RT buffer were from Gibco BRL (Cergy Pontoise-France). The promyelocytic leukemia cell line HL-60 was routinely cultured in suspension in RPMI supplemented with 10% fetal calf serum, glutamine, Hepes, buffer, penicillin, and streptomycin. The cells were maintained in exponential growth by dilution to 2 X 105/mlevery other day. A. Synthesis of Fluorescent Oligo(dT) All the solvents must be anhydrous. The 8% cetyltrimethylammonium bromide solution can be stored at 4"C, and must be warmed before use to facilitate dissolution. The FITC adsorbed on Celite and the dipyridyldisulfide (Aldrithiol) were stored at 4°C. The other reagents were stored desiccated at room temperature. The procedure has been described elsewhere (Godovikova et al., 1986). A total of 200 pg of 5' phosphorylated o(dT)(15 mer) was dissolved in 30 pl of distilled water and precipitated as the cetyltrimethylammonium salt by addition of 3 pl of 8% hexadecyltrimethylammonium bromide (CTAB). The precipitate was pelleted 5 min at 10,OOOg. CTAB (1.5 pl) was added and the suspension was centrifuged again. This stepwise addition of CTAB was repeated until no more precipitate was formed. At this stage the oligonucleotide was in its CTAB salt form, which is soluble in DMSO. The pellet was resuspended in dry methanol and evaporated to dryness in a vacuum drier (Speed Vac). The CTAB salt of o(dT) was dissolved in 200 pI of water-free DMSO containing N-methylimidazole (0.6 M ) , dipyridyldisulfide (0.3 M ) , and triphenylphosphine (0.3 M ) . The mixture was incubated for 15 min at room temperature to activate the terminal phosphate, and ethylenediamine was added (1.2 M final concentration) and incubated for a further 30 min. At this stage, the diamine was coupled to the 5' phosphate. The o(dT) derivative was precipitated by adding 1 ml of
5. Detection of m R N A by FCM
63
acetone containing 3% of LiCIO, and the precipitate was washed twice with acetone by centrifugation. The pellet was dried by evaporating the acetone at 60°C and was then dissolved in 100 p1 of 0.3 M triethylamine in water. The 5 ' terminal amino group was coupled to FITC by adding 2 mg of Celite containing 10% FITC and incubating the mixture overnight with constant agitation. The FITC-5'o(dT) was separated from the Celite by centrifugation and precipitated by adding 0. I volume of 3 M sodium acetate, pH 5, and 2 volumes of ethanol. After 1 hr at -20°C the precipitate was recovered by centrifugation for 15 min at 12,OOOg, washed with 70% ethanol, and dissolved in TBE buffer. The fluorescent oligonucleotide was purified from the uncoupled reaction products by electrophoresis on a 20% acrylamide sequencing gel. The brightly fluorescent band corresponding to the FITC-o(dT),, was excised, the acrylamide gel was crushed, and the FITC-o(dT) was eluted overnight with 0.5 ml of 0.1% Triton X-100, 0.3 M LiCIO, in H,O. The FITC-o(dT) was precipitated by addition of 3 ml of acetone containing 3% LiCIO, ( I hr at -2O"C), washed with acetone, dried, and dissolved in 200 pl of H,O. The concentration can be measured by spectrofluorometry, using dilutions of FITC as standard. B. Cell Fixation
FISH: 4 X lo6 HL-60 cells were washed in cold PBS and resuspended in 1 ml of 1% paraformaldehyde in PBS for 5 min at room temperature. After centrifugation (10 min, 200g, P C ) , the cells were resuspended in I ml of PBS, and 2.3 ml of absolute ethanol was added. After centrifugation for 3 min at IOOOg, the cells were resuspended in I ml of 70% ethanol and conserved at -20°C for several weeks. PRINS: prefixation with PFA was found to significantly reduce elongation of the primer by the reverse transcriptase. The samples for PRINS labeling were the fixed at a concentration of 4 x lo6 cells/ml with 70% ethanol. C. RNase Treatment
In some experiments, 4 x lo5 fixed cells were pelleted for 20 sec at 12,OOOg, resuspended in PBS containing 10 U/ml of RNase A, and incubated for 30 min at 37°C. The cells were then pelleted and conserved in 70% ethanol.
IV. Staining Procedures All the following manipulations were carried out in autoclaved conical microtubes; the centrifugations were for 30 sec at 12,OOOg in a microfuge; all solutions were sterilized by filtration at 0.22 pm and prepared with DEPC-treated distilled water.
64
Francis Belloc and Franqoise Durrieu
A. In Situ Hybridization with FITC-o(dT)
Add 3 p1 of a 10% solution of DEPC in ethanol to 50 pi ( 2 x lo5) of the fixed cell suspension. Incubate for 5 rnin at room temperature to inhibit endogenous RNase. Pellet the cells and wash with 100 pI of PBS containing 0.5% Tween 20 (PBST). Add 100 p15x SSPE (1 x SSPE: NaC1, 180 mM; EDTA, 1 mM; Na, HPO,, 5 mM) and leave to equilibrate for 1 hr at room temperature. Pellet the cells. Resuspend the cells in 10 pl of the hybridization mixture (HM): 0.1% SDS, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% albumin, 0.5 mg/ ml calf thymus DNA, and 0.5 pg/ml FITC-o(dT) in 5x SSPE. The mixture was incubated for 2 hr at 35°C under constant agitation. Add 40 pI of H 2 0 and incubate for 30 min at 35°C. Pellet the cells. Wash the cells in 100 pl of PBST; resuspend on 0.7 ml of PBST containing 1 pglml of propidium iodide and 1 Ulrnl of RNase. After 10 rnin at 20°C, the samples are ready for flow cytometry. B. PRINS Labeling with o(dT) as Primer
Pellet 100 pi of fixed cell suspension (4 X lo5 cells) and resuspend in 10 pl H20. Add 1 p1 of o(dT). Incubate for 10 rnin at 70"C, and cool on ice. Add 4 pI of 5 x RT buffer; 2 pI of 0.1 M DTT; 2pl of a mixture of 1 mM of each dGTP, dATP, and dCTP; 0.7 p1 of 1 mM fluorescein-12-dUTP; and 1 pl (200 U) of reverse transcriptase. Incubate for 60 rnin at 37°C. Add 500 pI of PBST. Pellet the cells. Resuspend the cells in 0.7 ml of PBST containing 1 pg/ml of propidium iodide and 1 U/ml of RNase. After 10 rnin at 20"C, the samples are ready for flow cytometry.
V. Critical Aspects of the Procedure and Controls Avoidance of any contamination with RNase is crucial to the success of any work concerning mRNA. The operator must wear gloves throughout the experiments. All the conical microfuge tubes and pipette tips must be autoclaved and kept separate from the rest of the laboratory supply. All the solutions were prepared using DEPC-treated distilled water. The duration of the hybridization step depends on the accessibility of the RNA to the probe and may vary from one experiment to another. It can be affected by the nature of the cells, the size of the oligonucleotide, and the fixation procedure, but 1 hr will usually be sufficient.
5. Detection of mRNA by FCM
65 Table I Verification of Specificity of Fish for Poly(A) ~
+
RNA
~
Sample
Ratio
%
Positive (0.5 pg/ml FITC-o(dT) Negative (no probe) FITC-o(dT) + poly(U)(3 mg/ml) FITC-o(dT) + poly(A)(3 rng/ml) Fixed cells treated with RNase Cells incubated with 1 u d m l Act-D for 6 hr.
3.7 I I .9
100
1.1
1.5 2. I
0 33 4 19 39
Note. The ratio was calculated as the mean fluorescence channel of the sample divided by the mean fluorescence channel of the negative control.
On counterstaining DNA with propidium iodide (PI), very low concentrations of PI must be used (1 to 3 pg/ml are enough to obtain a “cell-cycle shape” on the histogram) to avoid an excessive contamination of the faint green fluorescence by PI fluorescence. The specificity of the label must be assessed by running controls. For FISH (Table I), the specific labeling is sensitive to pretreatment of the cells with RNase; its intensity was significantly decreased by competitive unlabeled probes [o(dT) or poly(U)] and by competitive target [soluble poly(A)]. There was a significant decrease in labeling after incubating the cells with I ,ug/ml of D-actinomycin for 6 hr, showing the accuracy of the method for detecting the effect of drugs on mRNA metabolism. For PRINS (Table II), the labeling was sensitive to pretreatment with RNase and was dependent on the presence of both primer [o(dT)] and polymerase (reverse transcriptase). Excess TTP reduced the labeling, probably by competing with fluorescent dUTP for the transcriptase. Table I1 Verification of Specificity of Prins Sample
Ratio
%
Complete reaction mix Without reverse transcriptase Without o(dT) + 70 pM TTP Fixed cells treated with RNase Cells incubated with I pg/ml Act-D for 6 hr.
4. I
100 0
I 2 2.4 1.8 2.3
32 38 25 44
Note. The ratio was calculated as the mean fluorescence channel
of the sample divided by the mean fluorescence channel of the negative control.
Francis Belloc and Franqoise Durrieu
66
Incubation of the cells with D-actinomycin induced a decrease similar to that observed with the FISH method. For both methods, incubation of the cells with RNase after the labeling step will not significantly affect the fluorescent signal as the labeling (either hybridization or primer elongation) produces an RNase-resistant RNA/DNA hybrid.
VI. Instruments and FCM Analysis For FCM analysis, we used an ATC 3000 cytometer (ODAM-Brucker; Wissembourg, France) equipped with a 2025 Spectraphysics argon ion laser. The beam was tuned to emit 500 mW at 488 nm. The emission was split into green and red fluorescences by a 600-nm short-pass filter (Melles-Griot, Arnhem, Holland). The mRNA-specific green fluorescence (FITC) was collected through a 530 ? 30 nm band pass filter (Oriel, Paris, France). The DNA-specific red fluorescence (PI) was collected through a 600-nm long-pass filter (Melles-Griot). For each experiment, a sample of RNase-treated control cells was labeled and analyzed and the green signal was corrected for red contamination by electronic compensation until the biparametric FITC/PI histogram assumed a horizontal shape (Fig. 2). All further analyses were carried out at this electronic setting. The mRNA content of cells within specific regions of the cell cycle can be determined by gating the signal. For example, a gate can be set on the DNA histogram to provide the mRNA content of G,M cells before and after treatment with anthracycline. The data are collected and stored in list-mode fashion. and
i
4
0
Fig. 2
C
OM
FCM gated analysis of mRNA content on HL-60 cells. FISH using FITC-o(dT) was performed on control cells (A), on cells digested with RNase (B), and on cells treated with 10 nM idarubicin for 24 hr (C). The cells were counterstained with I pg/ml of PI and analyzed by FCM. The red fluorescence (DNA) is on the horizontal axis and the green fluorescence (mRNA) on the vertical axis. The green fluorescence in B was electronically compensated to obtain a horizontal distribution. The samples in A and C were then analyzed with the same electronic settings. The box encompassing the G2M cells was used to gate the analysis of mRNA content in G2M cells. The mean fluorescence value of control (in A) and Ida-treated cells (in C) was corrected for the value of RNase-treated cells (in B) and could then be compared.
67
5. Detection of mRNA by FCM
the measurements can then be retrieved for cells in a given phase of the cell cycle. For accurate measurement of the effect of a drug on the mRNA content, the mean fluorescence channel of the sample was corrected for the value of the RNase-treated sample (RNase-sensitive fluorescence).
VII. Results and Discussion Particular species of mRNA can be detected and quantified relatively by FISH using FITC-o(dT) as a probe followed by FCM (Table I and Belloc et a / . , 1993a). The effect of drugs on mRNA metabolism can be evaluated by multiparametnc analysis with gating on DNA content (Fig. 3 and Belloc et al., 1993b). It can be seen in Fig. 2A that G2M cells contain more mRNA than G, cells. Moreover, the mRNA in G2M are probably different than those of the G, cells (Campan et al., 1992). By gated acquisition, the mRNA content of G2Mcells from idarubicine-treated cells (Fig. 2C) can be readily compared with the mRNA content of G,M control cells (Fig. 2A). The mRNA content was defined as the RNase-sensitive green fluorescence: the mean green fluorescence channel of the defined cell population minus the mean green fluorescence of the same population in the RNase-treated sample. Using this method, we were
E '0
10
20
30
40
50
Incubation time (hrs)
Fig. 3 Flow cytometric analysis of the effect of drugs on the mRNA content. (A) HL-60 cells were treated with 10 ng/ml of idarubicine (open symbols) or 500 ng/ml of actinomycine D (closed symbols) for different periods, fixed, and hybridized with FITC-o(dT). The RNase-sensitive mRNA content of G2M cells was measured by flow cytometry as described in Fig. 2. The results are expressed as percent of initial mRNA content as a function of the duration of the incubation. (B) HL-60 cells were incubated for 8 hr with increasing concentrations of idarubicine. The RNase sensitive mRNA content of G,M cells was plotted as a function of the drug concentration.
68
Francis Belloc and Franqoise Durrieu
able to detect an accumulation of mRNA in Idarubicine-treated HL-60 cells and a decrease in mRNA in actinomycine-D-treated cells (Fig. 3 and Belloc et al., 1993a,b). The tedious and time consuming synthesis of FITC-o(dT) is the main drawback of this method, although it is relatively inexpensive. Much time could be saved by replacing the chemical coupling method with an enzymatic tailing of the o(dT) using terminal deoxynucleotidyl transferase and commercially available fluorescent deoxynucleotide triphosphates. The main drawback of such an enzymatic procedure would be difficulty in controlling the reaction to obtain probes that are homogeneous in size and fluorescence. Dideoxy fluorescent nucleotides triphosphates can be tried for this purpose. The PRINS labeling method using fluorescein-12dUTP as a label, o(dT) as a primer, and reverse transcriptase as a polymerase represents another approach, which in principle is an improvement on the preceding methods (Belund et al., 1991). Hybridization and fluorescence incorporation are performed in a single step; the primer confers the specificity, while the sensitivity is defined by the enzyme efficiency. In addition, primer molecules retained nonspecifically in the cytoplasm do not give rise to fluorescence incorporation in the absence of template, and several fluorescent molecules can be incorporated for each o(dT) molecule hybridized. Together these advantages should produce a very high signallnoise ratio. So far we have only obtained a signal to noise ratio of the same order of magnitude as that of FITC. Table I1 shows that fluorescence incorporation was RNase sensitive, was influenced by treatment of the cells with D-actinomycin, and was dependent on the presence of both o(dT) and reverse transcriptase, which is indicative of good specificity for poly(A)+ RNA. In practice, the PRINS method was found to be a rapid and sensitive way of detecting poly(A)+ RNA by FCM. However, relative quantification is only accurate if one operates within the linear part of the enzyme kinetics. If this condition is met, the incorporated fluorescence should be proportional to the amount of hybridized o(dT). References Bauman, J. G. J., and Bentvelzen, P. (1988). Cyromerry 9, 517-524. Bauman, J. G. J., Bayer, J. A., and van Dekken, H. (1990). J . Microsc. (Oxford) 157, 73-81. Bayer, J. A., and Bauman, J. G. J. (1990). Cyromerry 11, 132-143. Belloc, F., Lacombe, F., Dumain, P., Mergny, J. L., Lopez, F., Bernard, P.,Reiffers, J., and Boisseau, M. R. (1993a). Cyrometry 14, 339-343. Belloc, F., Lacombe, F., Dumain, P., Lopez, F., Bernard, P., Reiffers, J., and Boisseau, M. R. (1993b). Cytometry, Suppl. 6, 38. Bolund, L., Hindkjaer, J., Junker, S., Koch, J., Kolvraa, S., Mogensen, J., Nygaard, M., and Pedersen, S. (1991). Cyromerry, Suppl. 5,61. Campan, M., Desgranges, C., Gadeau, A. P., Millet, D., and Belloc, F. (1992). J . Cell. Physiol. 150,493-500. Darzynkiewicz, Z., Traganos, P., and Melamed, M. R. (1980). Cyromerry 1,98-108. Darzynkiewicz, Z., Kapuscinski, J., Traganos, F., and Crissman, H. (1987). Cyromerry 8,138-145. Godovikova, T. S . , Zarytova, V. F., and Khalimskaya, L. M. (1986). Bioorg. Khimi. U ,475-481.
5. Detection of mRNA by FCM
69
Jacobson, A. (1987). I n “Methods in Enzymology” (S. L. Berger and A. R . Kimmel, eds.), Vol. 152, pp. 254-261. Academic Press, San Diego. Lee, L. G . , Chen. C. H., and Chiuu, L. A. (1986). Cyromerry 7, 508-517. Morgensen, J . , and Kolvroa, S. (1991). Exp. Cell Res. 196, 92-98. Sage, B. H . , Jr., O’Connell, .I.P., and Mercouno, T. J . (1983). Cyfomefry 4, 222-227. Staiano-Coico, L . , Darzynkiewicz, Z.. and McMahon, C. K . (1989). Cell Tissue Kiner. 22,235-243.