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Enzymatic Amplification of Platelet-Specific Messenger RNA Using the Polymerase Chain Reaction

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Enzymatic Amplification of Platelet-Specific Messenger RNA Using the Polymerase Chain Reaction Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction. P J Newman, … , C J Cretney, R H Aster J Clin Invest. 1988;82(2):739-743. https://doi.org/10.1172/JCI113656. Research Article Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders. Find the latest version: https://jci.me/113656/pdf Rapid Publication Enzymatic Amplification of Platelet-specific Messenger RNA Using the Polymerase Chain Reaction Peter J. Newman,** Jack Gorski, * Gilbert C. White 11,I Susan Gidwitz,I Connie J. Cretney, * and Richard H. Aster* *The Blood Center ofSoutheastern Wisconsin, Milwaukee, Wisconsin 53233; tDepartment ofAnatomy and Cellular Biology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226; §Department ofMedicine, The University ofNorth Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and I1Departments ofMedicine and Pathology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Abstract from that of the analogue found in the nucleated cell from which the sequence was derived. Human platelets are derived from megakaryocytes as anu- It would be desirable to be able to study platelet-specific cleate cells, and thus contain only vestigial amounts of RNA gene sequences for a number of other reasons. Platelets express capable of being transcribed into protein. This has greatly a number of polymorphic epitopes on their surface that play hampered efforts to study directly platelet-specific gene prod- an important role in the etiology of alloimmune platelet dis- ucts and their associated polymorphisms. In this report, we orders (9, 10), and the molecular definition of alloantigenic describe direct amplification, using the polymerase chain reac- determinants would contribute to our understanding of the tion, of platelet-derived mRNA in amounts sufficient to permit morphological features of platelet antigens that are responsible detailed analysis, such as restriction mapping and nucleotide for eliciting an alloimmune response. These and other interest- sequencing. The ability to generate large amounts of cDNA ing platelet-related polymorphisms are often present in low from platelet-specific mRNA sequences should make possible frequency in the human population, making it nearly impossi- direct molecular characterization of normal platelet proteins, ble to predict or regulate the phenotypic makeup of a non- and facilitate the investigation of a wide variety of inherited platelet cDNA library. A preliminary report ( 11) suggests that platelet disorders. platelet mRNA can be prepared from individuals with myo- proliferative disorders in sufficient quantities to permit con- Introduction struction of a platelet-specific cDNA library, but it too would be limited in the number of polymorphic phenotypes repre- Human platelets contain more than 40 identifiable membrane sented. protein constituents, many of which have yet to be character- Recently, a method to enzymatically amplify short seg- ized. Since platelets are derived from megakaryocytes as non- ments of DNA has been developed (12-16). Known as the nucleated cells, it is generally believed that they possess only polymerase chain reaction (PcR),' this technique uses two vestigial amounts of protein-synthetic capability, although oligonucleotide primers to direct the repeated synthesis of specific protein biosynthesis in platelets has been demon- DNA bounded by their respective complementary sequences. strated ( 1-3 ). A primary deterrent to investigating the primary Since PcR has been reported to be capable of amplifying spe- structure of platelet-specific gene products has therefore been cific DNA sequences > 10 times, we decided to explore using the inability to obtain sufficient platelet mRNA to produce this method in the amplification of the vestigial amounts of cDNA libraries. A number of nucleated cell types have re- mRNA present in human platelets. In this report, we demon- cently been shown to synthesize proteins that are biochemi- strate that the RNA that can be derived from the platelets cally and immunologically related to their platelet counter- present in 50 ml of whole blood from normal individuals is parts (for a review, see reference 4), and cDNA libraries con- sufficient to produce microgram quantities of platelet-specific structed from these cells have been used to obtain the cDNA. This DNA is readily amenable to further detailed anal- nucleotide sequences of several platelet protein analogues ysis, including restriction mapping, subcloning, and nucleo- (5-8). Since these sequences are deduced from cDNA clones tide sequencing. derived from either transformed cell lines or cells of nonmega- karyocytic lineage, the possibility remains that the protein se- Methods quence of the actual platelet counterpart differs somewhat Materials. Taq DNA polymerase isolated from Thermus aquaticus Address reprint requests to Dr. Peter J. Newman, The Blood Center of was purchased from Perkin-Elmer Corp., Instrument Div., Norwalk, Southeastern Wisconsin, 1701 West Wisconsin Avenue, Milwaukee, CT. PGE,, Sarkosyl, and Antifoam A were obtained from Sigma WI 53233. Chemical Co., St. Louis, MO. Agarose used for preparative electropho- Received for publication 15 February 1988 and in revised form 7 resis was GTG-grade supplied by FMC Corp. Bioproducts, Rockland, April 1988. ME. Placental RNAse inhibitor (RNAsin) was purchased from Pro- mega Biotec, Madison, WI. Guanidinium isothiocyanate and cesium J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/88/08/0739/05 $2.00 1. Abbreviations used in this paper: GP, glycoprotein; PcR, polymer- Volume 82, August 1988, 739-743 ase chain reaction; PRP, platelet-rich plasma. Enzymatic Amplification ofPlatelet Messenger RNA 739 chloride were purchased from Bethesda Research Laboratories, Gaith- formed directly in this tube using cloned M-MLV reverse transcriptase ersburg, MD. Southern blot hybridization was performed after transfer and a cDNA synthesis kit (Bethesda Research Laboratories) according to Genescreen Plus nylon membranes (Dupont Co., Diagnostic and to the manufacturer's directions, except that the oligonucleotide BioResearch Systems, Wilmington, DE). All other chemicals used primer PcR-2 was substituted for Oligo (dT)12-18 to direct only were of reagent grade. GPIIIa-specific first strand synthesis. The first strand reaction mix Isolation of platelet RNA. Human platelets were isolated from contained 50 mmol/liter Tris pH 8.3, 75 mmol/liter KCI, 3 mmol/ whole blood as previously described (17). Briefly, 50 ml of venous liter MgCl2, 10 mmol/liter DTT, 0.5 mmol/liter each of dATP, blood collected from healthy volunteer donors was anticoagulated with dGTP, dCTP, and dTTP, 1.0 Mmol/liter PcR-2 oligonucleotide, and 7 ml of acid citrate dextrose (National Institutes of Health, Formula 500 U M-MLV reverse transcriptase, in a final volume of 50 Ml. cDNA A), and PGEI was added to a final concentration of 50 nmol/liter. synthesis was carried out at 370C for 60 min, at which time the reac- Platelet-rich plasma (PRP) was prepared by differential centrifugation, tion was stopped by chilling to 0C. The first strand reaction mix was and the platelets present in the upper half of the PRP were harvested diluted with an equal volume of PcR dilution buffer (25 mmol/liter and washed an additional two times in wash buffer (100 mmol/liter KCI, 1 umol/liter PcR2 oligonucleotide, 2 Mmol/liter PcR-I oligonu- NaCl, 8.5 mmol/liter Tris, 8.5 mmol/liter glucose, and 1 mmol/liter cleotide, and 0.02% gelatin) and heated to 94°C for 2 min to separate Na2EDTA, pH 7.4). By carefully avoiding the lower half of the PRP, the strands. Samples were allowed to cool to 37°C, at which time 1 Al we were consistently able to achieve a platelet/leukocyte ratio of at (5 U) of Taq polymerase was added to initiate the polymerase chain least 3000:1. The final platelet pellet was solubilized in 3.3 ml of buffer reaction. Amplification was performed for 30 cycles, with 10-min composed of 4 mol/liter guanidinium isothiocyanate, 5 mmol/liter primer extensions at 72°C, denaturation for 1 min 20 s at 94°C, and sodium citrate, 0.1 mol/liter 2-mercaptoethanol, 0.5% Sarkosyl, 0.1% primer annealings for 3 min 20 s at 37°C. An additional 2.5 U of Taq Antifoam A, and 40 U/ml placental RNAse inhibitor, layered onto a polymerase was added after cycles 10 and 20. After the PcR, the am- 1.2-ml cushion of 5.7 mol/liter CsCl, and centrifuged at 20'C in a plified samples were stored at -20°C. TiSO. 1 rotor at 35,000 rpm for 16 h (Beckman Instruments, Inc. Ful- Analysis of PcR reaction products. 10-15 Ml of each 100-Mul PcR lerton, CA). The nearly invisible RNA pellet was resuspended in reaction was analyzed on a 1% agarose gel, followed by staining with buffer made up of 10 mmol/ liter Tris, 5 mmol/ liter EDTA, and 1% ethidium bromide. Positive identification of the resulting DNA bands SDS, pH 7.4), extracted with chloroform/butanol, and ethanol precip- as authentic derivatives of the mRNA encoding GPIIIa was obtained itated essentially as described by Maniatis et al. (18). The resulting by Southern blot analysis, using a cloned GPIIIa insert that runs from purified total platelet RNA fraction recovered using this technique was base 45 of the published sequence ( 5 ) to the internal Eco RI site at base <1 gg per individual, and was used in all subsequent steps without 2,274.
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