THROMBOSIS RESEARCH 62; 127-135,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
QUANTITATIVE
ISOLATION OF RNA FROM HUMAN PLATELETS.
Isabelle Djaffar*, Didier Vilette*, Paul F. Bray", and Jean-Philippe Rosa* *U 150 INSERM, Hspital Lariboisiere, 75010 Paris, France ODepartment of Medicine, University of California, San Francisco, CA 94143, USA
;
(Received 11.7.1990; accepted in revised form 12.12.1990 by Editor M.C. Boffa) ABSTRACT We have adapted the acid-guanidinium-phenol-chloroform extraction rapid efficient of Chomczinsky and Sacchi to achieve procedure recovery of total RNA from human platelets. Sufficient platelet RNA (20 ug of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot anal sis K onors and detect the mRNAs for glycoproteins IIb on individual (GP 9 IIb) and IIIa (GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our Northern blotting and PCR, and simultaneous technique allows should be of great help to the characterization of therefore inherited platelet disorders such as Glanzmann's thrombasthenia.
INTRODUCTION Identification of platelet membrane proteins responsible for adhesion and basis of aggregation has led to a better understanding of the molecular inherited platelet abnormalities (1). The characterization of the underlying gene defects could benefit from analysis of the corresponding mRNAs : mRNAs can be altered quantitatively (usually decreased) or qualitatively (abnormal size, sequence alterations). Isolation of RNA from circulating megakaryocytic stem cells has been achieved in chronic myelogenic leukemia patients (2), but the technique used may not be easily transposable to patients with normal numbers of circulating stem cells. Isolation of platelet RNA thus represents an attractive alternative approach. Already suspected because platelets do synthesize proteins (3,4), the existence of platelet mRNA was further established because several platelet-specific mRNAs were demonstrated by either cDNA cloning (5,6), amplification by PCR (7), or Northern blot analysis (8). However these studies
Key words
: GP IIb-IIIa,
PCR, Northern blot.
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were based on methods which either required large quantities of blood (5,6,8), or were time-consuming (5,6,7), or yielded small amounts of RNA (7), and were clearly not adapted to routine platelet RNA isolation in individual donors. We now describe a rapid method adapted from that of Chomczinsky and Sacchi (10) for the quantitative isolation of undegraded total platele RNA. & From as little as 20 ml of whole blood, 1 to 3 ug of total RNA per 10 platelets was obtained. We also show that platelets contain undegraded ribosomal RNAs as well as intact messenger RNAs for GP IIb and GP IIIa. Using our method, efficient recovery of platelet RNA from single individuals is now achievable, and should facilitate the characterization of inherited platelet disorders, the analysis of platelet protein polymorphism or the isolation of new platelet-specific cDNAs. METHODS Materials: Formamide, phenol and guanidinium isothiocyanate were purchased from Bethesda Research Laboratories, Gaithersburg, MD. Glyoxal and Sarcosyl were obtained from Sigma Chemical Co., St. Louis, MO. 2-mercaptoethanol was from Biorad, Richmond, CA, and nitrocellulose membranes were from Millipore, Bedford, MA. All other chemicals were of reagent grade. Purification of platelet RNA: Routine precautions to prevent RNase contamination were performed as outlined by Maniatis et al (9). RNA extraction from whole platelet pellets was extracted by the acid-guanidinium-phenol-chloroform method of Chomczinsky and Sacchi (10) with the following modifications : briefly, human platelets were isolated either from whole blood units anticoagulated with sodium citrate/citric acid or from whole blood anticoagulated with 5 mM EDTA. Platelet-rich plasma (PRP) was obtained by centrifugation of the whole blood at 120 g for 15 min at 4OC. For PRP collection, care was taken not to perturb the interface with the red cell pellet to minimize contamination with white cells. Platelets were finally pelleted by centrifugation for 20 min at 1200 g and resuspended in denaturing buffer (4 M guanidinium isothiocyanate, 25 mM sodium citrate pH 7, 0.5 % sarcosyl, 0.1 M 2-mercaptoethanol). Phenol extraction was then performed as in reference 10 except that 1.5 ml microfuge tubes were used and siliconized ones for isopropanol precipitation. RNA was pelleted at 13 OOOg for 30 min at room temperature, and redissolved in l/10 of the original volume of denaturing buffer. Like in reference 10, a second round of isopropanol precipitation was carried out and the final RNA pellet was washed with 75% ethanol, quickly dissolved in diethylpyrocarbonate-treated water and either used immediately or stored frozen at -8OV. The amount of recovered RNA was determined by the absorption at 260 nm, using a ratio of 40 ug/ml per OD unit, and purity monitored by A260/A280 ratios. Cell counts: Platelets were counted in PRP in a hemocytometer by phase microscopy. Total white cell counts were assessed both manually in parallel with platelet counts, using the same hemocytometer, and in some experiments using an automatic cell counter (Coultertronics). Isolation of RNA from lymphocytes: Mononuclear cells were first separated by density gradient centrifugation (11). Nonadherent mononuclear cells were
LGP IIb : glycoprotein IIb ; GP IIIa : glycoprotein IIIa PCR : polymerase chain reaction ; PRP : platelet rich plasma
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resuspe ded in the guanidinium-containing denaturing buffer at a concentration 2 of 5x10 cells per 100 ul of buffer, and RNA extracted as described above. Northern blot analysis: Total RNA was denatured by glyoxal, subjected to electrophoresis to nitrocellulose, in a 1.1% agarose gel and transferred according to Thomas (12), except that baked filters were treated with 20 mM Tris pH 8.0 for 10 min. Hybridization and washings were carried out using routine procedures as described (12). cDNA probes: A 3.4kb full length cDNA for GP IIb was isolated from an HEL cell cDNA library (13). The GP IIb insert was subcloned into a pBluescript S/K+ plasmid (Stratagene, San Diego, CA) prior to its isolation. An Eco RI fragment of a GP IIIa cDNA representing the 2.2 kb most 5' sequences of the 6.2kb GP IIIa mRNA (14) was also subcloned into a pBluescript plasmid. Both GP IIb and GP IIIa inserts were then purified and used as probes. Probes were randomlabeled using a commercial kit (Random Priming Kit from Boehringer-Mannheim, Ingelheim, RFA) and ccording to the supplier's instructions. Average specific 8 activities were 1x10 cpm/ug. (15) with some chain reaction: PCR was conducted as published Polymerase modifications. 24 mer oligonucleotides LG and LH representing respectively nucleotides 1109 to 1132 and 597 to 621 of the GP IIb cDNA sequence (16) were LG and LH generate a 535 bp-long used as 3' and 5' primers respectively. fragment. LG and LH correspond to exons separated by several kb in the GP IIb gene (17) such that the 535 bp fragment cannot originate from amplification of genomic GP IIb. Total RNA was first reverse transcribed. Briefly 100 ng of LG was added to 0.05 to 1 ug of total RNA and denatured at 75OC for 3 min. The reaction was conducted at 42OC using 10 units of reverse transcriptase (GIBCO-BRL, Gaithersburg). After chilling on ice, the reaction was diluted 5 fold in 50 mM Tris pH 8.3, 50 mM KU, 2.5 mM MgC12, 10 mM 2-mercaptoethanol, 250 ug/ml gelatin, and added 105 ng of 5'primer LH. PCR was then carried out PCR cycles in an Intelligent Heating Block (Hybaid, Teddington, U.K.). consisted of a 45 s. denaturation step at 93"C, a 1 min annealing reaction at cycles were 55OC and a 2 min elongation reaction at 70°C. 35 identical programmed except that the denaturation reaction in the first cycle was at addition of 2.5 U. of polymerase 99OC for 7 min followed by Taq (Amersham-France) in the annealing step, and the last cycle included a 7 min termination reaction at 7ooc. 40 Pg of GP IIb cDNA inserted into the pBluescript plasmid was used in control reactions in which the reverse transcription step was omitted. The 535 bp amplified product was visualized after a 3 % agarose gel electrophoresis (3/4 Nusieve-GTG and l/4 SeaKem (FMC, Rockland, ME)) stained in ethidium bromide. RESULTS In a preliminary attempt at isolating platelet RNA, in order to minimize platelet handling and RNA degradation, we decided to directly solubilize platelets after PRP witput washing. As illustrated in row 1 of Table I an RNA yield of 0.15 ug per 10 platelets was obtained. This yield was of the order of magnitude of previous reports (5, 7) and still too low to perform Northern blots on small blood samples. In an effort to still improve RNA recovery we kept blood drawing, PRP, and platelet collection identical but introduced two modifications: first we used 1.5 ml polypropylene microcentrifuge tubes (siliconized for isopropanol precipitation) instead of large 50 ml polycarbonate tubes for all steps, and second we solubilized platelets at a higher concentration, 5X108 cells/100 ul of lysis buffer instead of 1x108
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cells/100 u&. The resulting recovery rates improved nearly 10 fold, averaging 1 ug per 10 platelets (rows 2 to 10 of Table I). Reduction of starting sample sizes from 250 ml to 10 ml, did not affect RNA yields indicating that our method was adapted to the handling of small samples. Thus it was clear that the sole careful managing of sample handling could considerably increase RNA yield (donors 2 to 10 compared to donor 1). However TABLE I
Donor number
1 2 3 4 5 6 7 8 9 10
Total Platelet RNA Recovery From 10 Independent Donors. RNA Number of _ RNA Volume ield platelets X lo8 recovered of PRP B ug/lO platelets ml ug 235 14 242 160 190 10 180 250 200 240
600 21 160 310 276 17 a nd nda 300 300
88 23 432 273 237 30 400 376 270 400
0.15 1.1 2.7 0.88 0.86 1.7: nd a nd 0.9 1.33
a nd : not determined
we wondered if part of the differences in yields between reference 7 (1 ug from 50 ml of blood) and ours might be due to a considerably higher leukocyte contamination in our platelet preparations. Our average contamination was 1 leukocyte in 5000 + 500 platelets by automatic counting and 2500 f 500 under the microscope. These were usual values, and most importantly werewithin the STANDARD
FIG 1: Agarose gel electrophoresis of total platelet RNA 0.5 ug of total RNA from different cell sources was subjected to electrophoresis in 1.1% agarose in 10 mM sodium phosphate pH 7.4, after denaturation in glyoxal-DMSO at 50°C (11). RNA was detected by fluorescence of the ethidium bromide dye. Lanes 1-3 : 0.2, 0.5, 1 ug of calf liver 18s and 28s RNA (Pharmacia, Uppsala, Sweden); lane 4: HEL cell RNA ; lane 5 : platelet RNA ; lane 6 : leukocyte RNA. 28 S and 18 S ribosomal RNA bands are indicated.
-
28s
-
18s
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range of that reported in ref. 7. In addition we were unable to detect lymphocyte transcripts (IgG light chain transcripts) in our platelet RNA by Northern blot (not shown). Thus leukocyte contamination could not explain the large difference in RNA recoveries. Isolated platelet RNA was intact as demonstrated by the presence in the platelet sample of sharp 18s ribosomal bands comigrating with those from leukocytes and HEL cells (Fig. 1). No high molecular weight bands were visible consistent with absence of major DNA contamination. Using a standard RNA of RNA platelet possible to estimate known also concentration, it was concentration, which correlated with OD measurements. To demonstrate the presence of platelet-specific transcripts32P_w;be;;z; performed a Nothern blot analysis of total platelet RNA using cDNAs specific for GP IIb and GP IIIa. In Fig. 2 decreasing amounts Of platelet RNA from a normal donor were analyzed. Normal undegraded GP IIb and were detected. In GP IIIa mRNAs, 3.4 and 6.2 kb in size respectively, addition, both messages could be detected using as little as 10 ug of total platelet RNA, i.e. the equivalent of approximately 10 ml of blood.
FIG 2: Detection of GPIIb and GPIIIa blot analysis of mRNAs by Northern total platelet RNA 30 to 2 ug of total RNA from platelets of a single donor was subjected to Northern blot transfer. cDNAs for GP IIb and GP IIIa were random-labeled independently, then mixed in a 1 : 1 ratio and hybridized to transferred RNA. Autoradiogram was exposed for 30 hours. The relative positions of GPIIb and GPIIIa transcripts are indicated and differ from the migration of 18s and 28s ribosomal RNAs.
DIa
IIb
Total platelet RNA prepared as outlined in Materials & Methods was reverse transcribed and amplified by PCR. Fig. 3 demonstrates that a 535 bp fragment corresponding to nucleotides 597 to 1132 of the GP IIb mRNA was amplified from
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32 P-labeled GP IIb cDNA PCR product was GP IIb-specific since it hybridized to in Southern blotting (not shown) and did not originate from amplification of genomic DNA since primers LG and LH correspond to separate exons in the GP IIb gene (17). In experiments not shown here GP IIb and GP IIIa mRNAs could be amplified using as little as 50 ng of total platelet RNA.
FIG 3: Agarose gel electrophoresis of GP IIb mRNA amplified by PCR Total RNA was reverse transcribed using the GP IIb-specific 3' primer LG. The first strand cDNA was amplified by PCR (35 cycles) using the GP IIb-specific 5' primer LH. 10 ~1 of 100 ul total reaction were electrophoresed in agarose gel. Bands were visualized by ethidium bromide. MW STD: 2.5 ug of @X174 phage DNA Hae III restriction fragments; lane 1: PCR of 40 pg of GP IIb cDNAcontaining pBluescript plasmid; lane 2: PCR of 1 ug of genomic DNA ; lane 3: PCR of 100 ng HEL cell total RNA; lane 4: PCR of 100 ng HEL cell total RNA using 3' primer LX (bases 1115 to 1138); lane 5: PCR of 100 ng of total platelet RNA using 3' primer LG. Arrow indicates LG-LH 535 bp GP IIb mRNA amplified product. Lower molecular weight bands probably represent coamplified non-specific products.
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DISCUSSION Our aim was to propose a rapid, simple and efficient method for extraction of platelet RNA from blood samples of limited size, and which would enable investigators to gain information on platelet-specific transcripts such as GPIIb and GPIIIa mRNAs. To this end we optimized the method of Chomczinsky and platelet washings and solubilizi8g Sacchi (10). We found that avoiding concentration (5x10 instead of 1x10 ) platelets at high 100 ul per considerably improved the yields of platelet RNA. This improvement can clearly not be due to leukocyte RNA contamination since we obtained the same kind of yield reported by others when omitting our modifications. Thus, the systematic reduction of volumes and the use of Eppendorff tubes (siliconized prior the step) appeared critical for efficient recovery of isopropanol precipitation RNA. Combined together these improvements allowed us to rapidly isolate an average of 30 ug of RNA per 20 ml whole blood samples, which is sufficient for Northern blot analysis of platelet transcripts such as GP IIb and GP IIIa. In addition, 100 ng of this RNA, i.e. less than 1 ml of blood, is sufficient to perform multiple PCR on both GP IIb and GP IIIa mRNAs. Our method greatly improved (10 fold or more) RNA yields mentioned in not surprising because these previous reports was (5,7). Indeed this investigators used either ultracentrifugation on cesium chloride gradients (7), or lysis in guanidinium and lithium chloride (5), both methods likely to yield low amounts of RNA: cesium gradients give notoriously bad yields (9), 5 involved multiple steps, including and the method used in reference resuspensions in buffers containing no RNAse inhibitors, and the handling of large volumes of samples and buffers. The finding that platelet RNA was undegraded may be surprising since circulating platelets have an average half-life of 8 days (18) whereas the half-life of eukaryotic mRNA has a typical average value of 6 to 24 hours (19). One explanation for this apparent discrepancy is that the RNA detected originated from "young" platelets. This would be in agreement with the increased rate of protein synthesis of "young" platelets with short half-lives observed in immune thrombocytopenic purpura patients (3). The fact that mRNA for platelet proteins can be easily assessed both qualitatively and quantitatively starting from small quantities of blood should be of help for the study of congenital bleeding disorders such as Glanzmann's thrombasthenia. In this disease GPIIb and/or GPIIIa polypeptides, the two subunits of the fibrinogen receptor, are either decreased in quantity or functionally altered (for review see ref. 1). Some of the corresponding gene anomalies leading to alterations in the amount or size of GP IIb and/or GP IIIa transcripts could be assessed by Northern blotting with as little as 20 ml of blood. A fraction of the same blood sample could be subjected to PCR after reverse transcription of platelet RNA to rapidly identify sequence anomalies such as point mutations most likely to be involved in variants of thrombasthenia (20,21). ACKNOWLEDGMENTS This work was supported by grants from IVS, INSERM and CNRS. I.D. is a recipient of a Ministere de la Recherche et de la Technologie Award. D.V. is supported by a fellowship from IVS. P.F.B. is a recipient of Physician-Scientist Award HL01815 from the NIH. J.-P. R. is Charge de Recherche INSERM. We wish to thank Dr N. Kieffer who first introduced us to the method of Chomczynski and Sacchi, Dr. J.N. George for valuable advice in the preparation of this manuscript, Agnes Chasme for excellent secretarial assistance.
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REFERENCES 1.
GEORGE, J.N., NURDEN, A.T. and PHILLIPS, D.R. Molecular defects in interactions of platelets with the vessel wall. N. Engl. J. Med. 311, 1084-1098, 1984.
2.
UZAN, G., FRACHET, P., LAJMANOVICH, A., PRANDINI, M.-H., DENARIER, .E., DUPERRAY, A., LOFTUS, J., GINSBERG, M., PLOW, E. and MARGUERIE, G. cDNA clones for human platelet IIb corresponding to mRNA from megakaryocytes and HEL cells. Evidence for an extensive homology to other Arg-Gly-Asp adhesion receptors. Eur. J. Biochem. 171, 87-93, 1988.
3.
and KIEFFER, N., GUICHARD, J., FARCET, J.-P., VAINCHENKER, W. BRETON-GORIUS, J. Biosynthesis of major platelet proteins in human platelets.
4.
Eur. J. Biochem.
1987.
BELLOC, F., HEILMANN, E., COMBRIE, R., BOISSEAU, M.R. and NURDEN, A.T. Protein synthesis and storage in human platelets : a defective storage of fibrinogen in platelets in Glanzmann's thrombasthenia. Biochim. Biophys. Acta 925, 218-225,
5.
164, 189-195,
1987.
WICKI, A.N., WALZ, A., GERBER-HUBER, S., WENGER, R-H., VORNHAGEN, R. and CLEMETSON, K.J. Isolation and characterization of human blood platelet mRNA and construction of a cDNA library in lambda gt 11. Thromb. Haemost. 61, 448-453,
1989.
6.
WENGER, R.H., WICKI, A.N., WALZ, A., KIEFFER, N. and CLEMETSON, K.J. Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt 11 expression library. Blood 73, 1498-1503, 1989.
7.
NEWMAN, P.J., GORSKI, J., WHITE, G.C., GIDWITZ, S., CRETNEY, C.J. and ASTER, R.H. Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction. J. Clin. Invest. 82, 739-743, 1988.
8.
SOTTILE, J., MOSHER, D-F., FULLENWEIDER, J., GEORGE, J. N. Human platelets contain mRNA transcripts for platelet factor 4 and actin. Thromb. Haemost. 62, 1100-1102,
9.
MANIATIS, Laboratory
T.,
1989.
FRITSCH, E.F. and SAMBROOK, J. Molecular Cloning. A Cold Spring Harbor: Cold Spring Harbor Laboratory,
Manual.
1982. 10. CHOMCZINSKY, P. and SACCHI, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159, 1987.
11. BEVILACQUA, M.P., AMRANI, D., MOSESSON, M.W. and BIANCO, C. Receptors for cold-insoluble globulin (plasma fibronectin) on human monocytes. J. Exp. Med. i53, 42-60, 1981.
12. THOMAS, P.S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205, 1980.
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13. BRAY, P.F., BARSH, G., ROSA, J.-P., LUO, X.Y., MAGENLS, E., IIb chromosomal Platelet glycoprotein localization M.A. expression. Proc. Natl. Acad. Sci. 85, 8683-8687 1%?8. 14. ROSA, K.W., human Blood
and SHUMAN, tissue and
J.-P., BRAY, P.F., GAYET, O., JOHNSTON, G.I., COOK, R.G., JACKSON, SHUMAN, M.A. and MCEVER, R.P. Cloning of glycoprotein IIIa cDNA from erythroleukemia cells and localization of the gene to chromosome 17. 72, 593-600, 1988.
15. SAIKI, R.K., SCHARF, S., FALOONA, F., MULLIS, K.B., HORN, G.T., ERLICH, H.A. and ARNHEIM, N. Enzymatic amplification of beta-globin gene sequences and restriction site analysis for diagnosis of sickle cell anemia. Science (Wash. DC) 230, 1350-1354, 1985. 16. PONCZ, M., EISMAN, R., HEIDENREICH, R., SILVER, S.M., VILAIRE, G., SURREY, S ., SCHWARTZ, E. and BENNETT, J.S. Structure of the platelet membrane of the vitronectin and glycoprotein IIb. Homology to the alpha-subunits fibronectin membrane receptors. J. Biol. Chem. 262, 8476-8482, 1987. 17. HEIDENREICH, R., EISMAN, R., SURREY, S., DELGROSSO, of the SCHWARTZ, E. and PONCZ, M. Organization glycoprotein IIb. Biochemistry 29, 1232-44, 1990.
K., BENNETT, J.S., gene for platelet
of the life-span of 18. LEEKSMA, C.U.W. and COHEN, J.A. The determination human blood platelets using diisopropyl fluorophosphate. J. Clin. Invest. 35, 964-969, 1956. 19. LEWIN, B. Genes. New-York:
John Wiley and Sons, 1987, p. 762.
20. GINSBERG, M.-H., LIGHTSEY, A., KUNICKI, T.J., KAUFMANN, A., MARGUERIE, G., and PLOW, E.F. Divalent cation regulation of the surface orientation of platelet membrane glycoprotein IIb. Correlation with fibrinogen binding function and definition of a novel variant of Glanzmann's thrombasthenia. J. Clin. Invest. 78, 1103-1111, 1986. 21. NURDEN, A.T., ROSA, J.-P., FOURNIER, D., LEGRAND, C., DIDRY, D., PARQUET, A ., and PIDARD, D. A variant of Glanzmann's thrombasthenia with abnormal glycoprotein IIb-IIIa complexes in the platelet membrane. J. Clin. Invest. 79, 962-969, 1987.
QUANTITATIVE
ISOLATION OF RNA FROM HUMAN PLATELETS.
Isabelle Djaffar*, Didier Vilette*, Paul F. Bray", and Jean-Philippe Rosa* *U 150 INSERM, Hspital Lariboisiere, 75010 Paris, France ODepartment of Medicine, University of California, San Francisco, CA 94143, USA
;
(Received 11.7.1990; accepted in revised form 12.12.1990 by Editor M.C. Boffa) ABSTRACT We have adapted the acid-guanidinium-phenol-chloroform extraction rapid efficient of Chomczinsky and Sacchi to achieve procedure recovery of total RNA from human platelets. Sufficient platelet RNA (20 ug of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot anal sis K onors and detect the mRNAs for glycoproteins IIb on individual (GP 9 IIb) and IIIa (GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our Northern blotting and PCR, and simultaneous technique allows should be of great help to the characterization of therefore inherited platelet disorders such as Glanzmann's thrombasthenia.
INTRODUCTION Identification of platelet membrane proteins responsible for adhesion and basis of aggregation has led to a better understanding of the molecular inherited platelet abnormalities (1). The characterization of the underlying gene defects could benefit from analysis of the corresponding mRNAs : mRNAs can be altered quantitatively (usually decreased) or qualitatively (abnormal size, sequence alterations). Isolation of RNA from circulating megakaryocytic stem cells has been achieved in chronic myelogenic leukemia patients (2), but the technique used may not be easily transposable to patients with normal numbers of circulating stem cells. Isolation of platelet RNA thus represents an attractive alternative approach. Already suspected because platelets do synthesize proteins (3,4), the existence of platelet mRNA was further established because several platelet-specific mRNAs were demonstrated by either cDNA cloning (5,6), amplification by PCR (7), or Northern blot analysis (8). However these studies
Key words
: GP IIb-IIIa,
PCR, Northern blot.
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were based on methods which either required large quantities of blood (5,6,8), or were time-consuming (5,6,7), or yielded small amounts of RNA (7), and were clearly not adapted to routine platelet RNA isolation in individual donors. We now describe a rapid method adapted from that of Chomczinsky and Sacchi (10) for the quantitative isolation of undegraded total platele RNA. & From as little as 20 ml of whole blood, 1 to 3 ug of total RNA per 10 platelets was obtained. We also show that platelets contain undegraded ribosomal RNAs as well as intact messenger RNAs for GP IIb and GP IIIa. Using our method, efficient recovery of platelet RNA from single individuals is now achievable, and should facilitate the characterization of inherited platelet disorders, the analysis of platelet protein polymorphism or the isolation of new platelet-specific cDNAs. METHODS Materials: Formamide, phenol and guanidinium isothiocyanate were purchased from Bethesda Research Laboratories, Gaithersburg, MD. Glyoxal and Sarcosyl were obtained from Sigma Chemical Co., St. Louis, MO. 2-mercaptoethanol was from Biorad, Richmond, CA, and nitrocellulose membranes were from Millipore, Bedford, MA. All other chemicals were of reagent grade. Purification of platelet RNA: Routine precautions to prevent RNase contamination were performed as outlined by Maniatis et al (9). RNA extraction from whole platelet pellets was extracted by the acid-guanidinium-phenol-chloroform method of Chomczinsky and Sacchi (10) with the following modifications : briefly, human platelets were isolated either from whole blood units anticoagulated with sodium citrate/citric acid or from whole blood anticoagulated with 5 mM EDTA. Platelet-rich plasma (PRP) was obtained by centrifugation of the whole blood at 120 g for 15 min at 4OC. For PRP collection, care was taken not to perturb the interface with the red cell pellet to minimize contamination with white cells. Platelets were finally pelleted by centrifugation for 20 min at 1200 g and resuspended in denaturing buffer (4 M guanidinium isothiocyanate, 25 mM sodium citrate pH 7, 0.5 % sarcosyl, 0.1 M 2-mercaptoethanol). Phenol extraction was then performed as in reference 10 except that 1.5 ml microfuge tubes were used and siliconized ones for isopropanol precipitation. RNA was pelleted at 13 OOOg for 30 min at room temperature, and redissolved in l/10 of the original volume of denaturing buffer. Like in reference 10, a second round of isopropanol precipitation was carried out and the final RNA pellet was washed with 75% ethanol, quickly dissolved in diethylpyrocarbonate-treated water and either used immediately or stored frozen at -8OV. The amount of recovered RNA was determined by the absorption at 260 nm, using a ratio of 40 ug/ml per OD unit, and purity monitored by A260/A280 ratios. Cell counts: Platelets were counted in PRP in a hemocytometer by phase microscopy. Total white cell counts were assessed both manually in parallel with platelet counts, using the same hemocytometer, and in some experiments using an automatic cell counter (Coultertronics). Isolation of RNA from lymphocytes: Mononuclear cells were first separated by density gradient centrifugation (11). Nonadherent mononuclear cells were
LGP IIb : glycoprotein IIb ; GP IIIa : glycoprotein IIIa PCR : polymerase chain reaction ; PRP : platelet rich plasma
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resuspe ded in the guanidinium-containing denaturing buffer at a concentration 2 of 5x10 cells per 100 ul of buffer, and RNA extracted as described above. Northern blot analysis: Total RNA was denatured by glyoxal, subjected to electrophoresis to nitrocellulose, in a 1.1% agarose gel and transferred according to Thomas (12), except that baked filters were treated with 20 mM Tris pH 8.0 for 10 min. Hybridization and washings were carried out using routine procedures as described (12). cDNA probes: A 3.4kb full length cDNA for GP IIb was isolated from an HEL cell cDNA library (13). The GP IIb insert was subcloned into a pBluescript S/K+ plasmid (Stratagene, San Diego, CA) prior to its isolation. An Eco RI fragment of a GP IIIa cDNA representing the 2.2 kb most 5' sequences of the 6.2kb GP IIIa mRNA (14) was also subcloned into a pBluescript plasmid. Both GP IIb and GP IIIa inserts were then purified and used as probes. Probes were randomlabeled using a commercial kit (Random Priming Kit from Boehringer-Mannheim, Ingelheim, RFA) and ccording to the supplier's instructions. Average specific 8 activities were 1x10 cpm/ug. (15) with some chain reaction: PCR was conducted as published Polymerase modifications. 24 mer oligonucleotides LG and LH representing respectively nucleotides 1109 to 1132 and 597 to 621 of the GP IIb cDNA sequence (16) were LG and LH generate a 535 bp-long used as 3' and 5' primers respectively. fragment. LG and LH correspond to exons separated by several kb in the GP IIb gene (17) such that the 535 bp fragment cannot originate from amplification of genomic GP IIb. Total RNA was first reverse transcribed. Briefly 100 ng of LG was added to 0.05 to 1 ug of total RNA and denatured at 75OC for 3 min. The reaction was conducted at 42OC using 10 units of reverse transcriptase (GIBCO-BRL, Gaithersburg). After chilling on ice, the reaction was diluted 5 fold in 50 mM Tris pH 8.3, 50 mM KU, 2.5 mM MgC12, 10 mM 2-mercaptoethanol, 250 ug/ml gelatin, and added 105 ng of 5'primer LH. PCR was then carried out PCR cycles in an Intelligent Heating Block (Hybaid, Teddington, U.K.). consisted of a 45 s. denaturation step at 93"C, a 1 min annealing reaction at cycles were 55OC and a 2 min elongation reaction at 70°C. 35 identical programmed except that the denaturation reaction in the first cycle was at addition of 2.5 U. of polymerase 99OC for 7 min followed by Taq (Amersham-France) in the annealing step, and the last cycle included a 7 min termination reaction at 7ooc. 40 Pg of GP IIb cDNA inserted into the pBluescript plasmid was used in control reactions in which the reverse transcription step was omitted. The 535 bp amplified product was visualized after a 3 % agarose gel electrophoresis (3/4 Nusieve-GTG and l/4 SeaKem (FMC, Rockland, ME)) stained in ethidium bromide. RESULTS In a preliminary attempt at isolating platelet RNA, in order to minimize platelet handling and RNA degradation, we decided to directly solubilize platelets after PRP witput washing. As illustrated in row 1 of Table I an RNA yield of 0.15 ug per 10 platelets was obtained. This yield was of the order of magnitude of previous reports (5, 7) and still too low to perform Northern blots on small blood samples. In an effort to still improve RNA recovery we kept blood drawing, PRP, and platelet collection identical but introduced two modifications: first we used 1.5 ml polypropylene microcentrifuge tubes (siliconized for isopropanol precipitation) instead of large 50 ml polycarbonate tubes for all steps, and second we solubilized platelets at a higher concentration, 5X108 cells/100 ul of lysis buffer instead of 1x108
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cells/100 u&. The resulting recovery rates improved nearly 10 fold, averaging 1 ug per 10 platelets (rows 2 to 10 of Table I). Reduction of starting sample sizes from 250 ml to 10 ml, did not affect RNA yields indicating that our method was adapted to the handling of small samples. Thus it was clear that the sole careful managing of sample handling could considerably increase RNA yield (donors 2 to 10 compared to donor 1). However TABLE I
Donor number
1 2 3 4 5 6 7 8 9 10
Total Platelet RNA Recovery From 10 Independent Donors. RNA Number of _ RNA Volume ield platelets X lo8 recovered of PRP B ug/lO platelets ml ug 235 14 242 160 190 10 180 250 200 240
600 21 160 310 276 17 a nd nda 300 300
88 23 432 273 237 30 400 376 270 400
0.15 1.1 2.7 0.88 0.86 1.7: nd a nd 0.9 1.33
a nd : not determined
we wondered if part of the differences in yields between reference 7 (1 ug from 50 ml of blood) and ours might be due to a considerably higher leukocyte contamination in our platelet preparations. Our average contamination was 1 leukocyte in 5000 + 500 platelets by automatic counting and 2500 f 500 under the microscope. These were usual values, and most importantly werewithin the STANDARD
FIG 1: Agarose gel electrophoresis of total platelet RNA 0.5 ug of total RNA from different cell sources was subjected to electrophoresis in 1.1% agarose in 10 mM sodium phosphate pH 7.4, after denaturation in glyoxal-DMSO at 50°C (11). RNA was detected by fluorescence of the ethidium bromide dye. Lanes 1-3 : 0.2, 0.5, 1 ug of calf liver 18s and 28s RNA (Pharmacia, Uppsala, Sweden); lane 4: HEL cell RNA ; lane 5 : platelet RNA ; lane 6 : leukocyte RNA. 28 S and 18 S ribosomal RNA bands are indicated.
-
28s
-
18s
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range of that reported in ref. 7. In addition we were unable to detect lymphocyte transcripts (IgG light chain transcripts) in our platelet RNA by Northern blot (not shown). Thus leukocyte contamination could not explain the large difference in RNA recoveries. Isolated platelet RNA was intact as demonstrated by the presence in the platelet sample of sharp 18s ribosomal bands comigrating with those from leukocytes and HEL cells (Fig. 1). No high molecular weight bands were visible consistent with absence of major DNA contamination. Using a standard RNA of RNA platelet possible to estimate known also concentration, it was concentration, which correlated with OD measurements. To demonstrate the presence of platelet-specific transcripts32P_w;be;;z; performed a Nothern blot analysis of total platelet RNA using cDNAs specific for GP IIb and GP IIIa. In Fig. 2 decreasing amounts Of platelet RNA from a normal donor were analyzed. Normal undegraded GP IIb and were detected. In GP IIIa mRNAs, 3.4 and 6.2 kb in size respectively, addition, both messages could be detected using as little as 10 ug of total platelet RNA, i.e. the equivalent of approximately 10 ml of blood.
FIG 2: Detection of GPIIb and GPIIIa blot analysis of mRNAs by Northern total platelet RNA 30 to 2 ug of total RNA from platelets of a single donor was subjected to Northern blot transfer. cDNAs for GP IIb and GP IIIa were random-labeled independently, then mixed in a 1 : 1 ratio and hybridized to transferred RNA. Autoradiogram was exposed for 30 hours. The relative positions of GPIIb and GPIIIa transcripts are indicated and differ from the migration of 18s and 28s ribosomal RNAs.
DIa
IIb
Total platelet RNA prepared as outlined in Materials & Methods was reverse transcribed and amplified by PCR. Fig. 3 demonstrates that a 535 bp fragment corresponding to nucleotides 597 to 1132 of the GP IIb mRNA was amplified from
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32 P-labeled GP IIb cDNA PCR product was GP IIb-specific since it hybridized to in Southern blotting (not shown) and did not originate from amplification of genomic DNA since primers LG and LH correspond to separate exons in the GP IIb gene (17). In experiments not shown here GP IIb and GP IIIa mRNAs could be amplified using as little as 50 ng of total platelet RNA.
FIG 3: Agarose gel electrophoresis of GP IIb mRNA amplified by PCR Total RNA was reverse transcribed using the GP IIb-specific 3' primer LG. The first strand cDNA was amplified by PCR (35 cycles) using the GP IIb-specific 5' primer LH. 10 ~1 of 100 ul total reaction were electrophoresed in agarose gel. Bands were visualized by ethidium bromide. MW STD: 2.5 ug of @X174 phage DNA Hae III restriction fragments; lane 1: PCR of 40 pg of GP IIb cDNAcontaining pBluescript plasmid; lane 2: PCR of 1 ug of genomic DNA ; lane 3: PCR of 100 ng HEL cell total RNA; lane 4: PCR of 100 ng HEL cell total RNA using 3' primer LX (bases 1115 to 1138); lane 5: PCR of 100 ng of total platelet RNA using 3' primer LG. Arrow indicates LG-LH 535 bp GP IIb mRNA amplified product. Lower molecular weight bands probably represent coamplified non-specific products.
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DISCUSSION Our aim was to propose a rapid, simple and efficient method for extraction of platelet RNA from blood samples of limited size, and which would enable investigators to gain information on platelet-specific transcripts such as GPIIb and GPIIIa mRNAs. To this end we optimized the method of Chomczinsky and platelet washings and solubilizi8g Sacchi (10). We found that avoiding concentration (5x10 instead of 1x10 ) platelets at high 100 ul per considerably improved the yields of platelet RNA. This improvement can clearly not be due to leukocyte RNA contamination since we obtained the same kind of yield reported by others when omitting our modifications. Thus, the systematic reduction of volumes and the use of Eppendorff tubes (siliconized prior the step) appeared critical for efficient recovery of isopropanol precipitation RNA. Combined together these improvements allowed us to rapidly isolate an average of 30 ug of RNA per 20 ml whole blood samples, which is sufficient for Northern blot analysis of platelet transcripts such as GP IIb and GP IIIa. In addition, 100 ng of this RNA, i.e. less than 1 ml of blood, is sufficient to perform multiple PCR on both GP IIb and GP IIIa mRNAs. Our method greatly improved (10 fold or more) RNA yields mentioned in not surprising because these previous reports was (5,7). Indeed this investigators used either ultracentrifugation on cesium chloride gradients (7), or lysis in guanidinium and lithium chloride (5), both methods likely to yield low amounts of RNA: cesium gradients give notoriously bad yields (9), 5 involved multiple steps, including and the method used in reference resuspensions in buffers containing no RNAse inhibitors, and the handling of large volumes of samples and buffers. The finding that platelet RNA was undegraded may be surprising since circulating platelets have an average half-life of 8 days (18) whereas the half-life of eukaryotic mRNA has a typical average value of 6 to 24 hours (19). One explanation for this apparent discrepancy is that the RNA detected originated from "young" platelets. This would be in agreement with the increased rate of protein synthesis of "young" platelets with short half-lives observed in immune thrombocytopenic purpura patients (3). The fact that mRNA for platelet proteins can be easily assessed both qualitatively and quantitatively starting from small quantities of blood should be of help for the study of congenital bleeding disorders such as Glanzmann's thrombasthenia. In this disease GPIIb and/or GPIIIa polypeptides, the two subunits of the fibrinogen receptor, are either decreased in quantity or functionally altered (for review see ref. 1). Some of the corresponding gene anomalies leading to alterations in the amount or size of GP IIb and/or GP IIIa transcripts could be assessed by Northern blotting with as little as 20 ml of blood. A fraction of the same blood sample could be subjected to PCR after reverse transcription of platelet RNA to rapidly identify sequence anomalies such as point mutations most likely to be involved in variants of thrombasthenia (20,21). ACKNOWLEDGMENTS This work was supported by grants from IVS, INSERM and CNRS. I.D. is a recipient of a Ministere de la Recherche et de la Technologie Award. D.V. is supported by a fellowship from IVS. P.F.B. is a recipient of Physician-Scientist Award HL01815 from the NIH. J.-P. R. is Charge de Recherche INSERM. We wish to thank Dr N. Kieffer who first introduced us to the method of Chomczynski and Sacchi, Dr. J.N. George for valuable advice in the preparation of this manuscript, Agnes Chasme for excellent secretarial assistance.
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