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Absolute Quantification of Human Chorionic Gonadotropin-Beta mRNA with TaqMan Detection

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Absolute Quantification of Human Chorionic Gonadotropin-Beta mRNA with TaqMan Detection
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  Human Chorionic Gonadotropin-  β   mRNA47  M  OLECULAR   B  IOTECHNOLOGY  Volume 14, 2000    RESEARCH 47 Absolute Quantification of Human Chorionic Gonadotropin- β mRNA with TaqMan™ Detection T. Reimer, 1,*   D. Koczan, 2   V. Briese, 1 K. Friese, 1  D. Richter, 1  H.J. Thiesen, 2   and U. Jeschke 1 Molecular Biotechnology   1999 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2000/14:1/47–57/$12.75 *Author to whom all correspondence and reprint requests should be addressed. Universitäts-Frauenklinik Doberaner Str. 142 D-18057Rostock, Germany, E-mail: toralf.reimer@med.uni-rostock.de 1 Department of Obstetrics and Gynaecology 2 Institute of Immunology, Fac-ulty of Medicine, University of Rostock, Germany 1. Introduction The human chorionic gonadotropin- β  (HCG β )subunit is encoded by a multigene cluster on chro-mosome 19 composed of six homologous se-quences (genes or pseudogenes). They areprimarily distinguished by sequences in the 5'nontranslated region of the first exon. It seemslikely that all these genes are transcribed, al-though most of the mRNAs in vivo are transcriptsfrom genes 5, 3, and 8 (1) . Only genes 5, 3, 8, and7 present the open reading frame of the HCG β subunit protein. The other two genes 1 and 2 areconsidered pseudogenes because they contain aseveral hundred base pairs (bp) insertion that re-places the promoter and most of the 5'untranslated region (2) .Because HCG is a placental protein hormone,its presence in nonpregnant subjects indicates anectopic secretion. HCG β  mRNAs were detectedin malignant cells but also in normalnontrophoblastic cells (3) . Several previous stud-ies have screened for unusual HCG β  expressionpattern of the individual genes 5, 3, 8, and 7 (4) .However, it is possible that a functional level of HCG β  subunit can be obtained by expression of various  β  genes and that it is the total level of HCG β  produced rather than the expression of individual β  genes that is important as a marker indifferent in vivo and in vitro conditions. For thisreason, we were interested in determining the in-dividual total amount of HCG β  mRNA.Because only methods for qualitative or semiquantitative evaluation of HCG β  mRNA lev- Abstract We describe a reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of humanchorionic gonadotropin- β  (HCG β ) mRNA copies using the TaqMan™ system. To evaluate our quantitativeassay, we analyzed HCG β  transcripts of all protein coding genes (HCG β  5, 3, 8, and 7) in human RNApanels of different normal tissues and in glycodelin–A–stimulated trophoblast cell cultures. Absolute quan-tification using HCG β  TaqMan probe was found to be highly reproducible. Our study of RNA panels con-firms recently published results that expression of HCG β  transcripts is a common feature of a great varietyof different normal tissues. High levels of HCG β  mRNAs (>1.000 molecules per 200 ng RNA) were de-tected in placenta, uterus, and testis. An increase of HCG β  mRNA expression (1.7-fold) was detected at 150 µ g/mL glycodelin-A treatment in trophoblast cell culture. Time-dependence study showed that the increasein HCG β  mRNA level was evident at 60 min after glycodelin-A treatment. In summary, we have developeda highly sensitive one-tube, one-enzyme quantitative RT-PCR system that is time-saving and avoids post-amplification procedures. Index Entries:  Glycodelin-A; human chorionic gonadotropin; quantitative RT–PCR; TaqMan; tropho-blast cell culture.  48 Reimer et al. M  OLECULAR   B  IOTECHNOLOGY  Volume 14, 2000  els have been available, a method that could de-tect the absolute mRNA copy number wouldgreatly improve the differentiation within thepositive samples and accurately define the cutoff values. However, many early reports of reversetranscriptase-polymerase chain reaction (RT-PCR) described quantification of the PCR prod-uct, but did not measure the initial target sequencequantity  (5) .The recent development of the 5' nucleaseassay for detecting PCR products uses anonextendable oligonucleotide hybridizationprobe  (6) . The probe is labeled with a reporter fluorescent dye (FAM [6-carboxy-fluorescein]) atthe 5' end and a quencher-fluorescent dye(TAMRA [6-carboxy-tetramethyl-rhodamine]) atthe 3' end. The probes are designed to hybridizewithin the target sequence to generate a signal thataccumulates during PCR cycling in proportion tothe accumulation of amplification products (7) .In this study, we used the ABI PRISM 7700Sequence Detector System (TaqMan™) and thefluorogenic probes of the Applied BiosystemsDivision, Perkin-Elmer (Foster City, CA). Toevaluate our specific HCG β  assay, we screened ahuman total RNA panel of 26 different tissuetypes for HCG β  mRNA copy numbers. Further-more, we confirmed our previous results aboutHCG protein stimulation by glycodelin-A (GdA)in trophoblast cell culture (8)  on the mRNA level. 2. Materials and Methods 2.1. RNA Extraction  We used commercially available human totalRNA panels of 26 normal (i.e., nondiseased) tis-sue types (Clontech, Palo Alto, CA) for humanbody HCG β  mRNA screening. These total RNAswere purified using a modified guanidiniumisothiocyanate method and were provided as anaqueous solution.Total RNA from cultured trophoblastic cellswas extracted by TRIzol® LS Reagent (LifeTechnologies, Grand Island, NY) according to themanufacturer’s protocol. RNA was purified fromculture dishes 30, 60, 90, 120, and 180 min after glycodelin-A stimulation. Purified RNA wasquantified and evaluated for purity by UV spec-trophotometry. To further assess the quality of RNA, all specimens were tested by analysis of housekeeping gene expression (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) usingconventional RT-PCR. 2.2. Primer and Probe Design  HCG β  primers and probe were designed usingthe Primer Express 1.0 program (PE AppliedBiosystems, Foster City, CA). Fig. 1  shows thenucleotide sequences for the oligonucleotide hy-bridization probe and primers. The reverse primer was designed to span an exon/intron junction toavoid amplification of DNA sequences, whereasthe forward primer was complementary to the 3'end of exon 1. Primers and probe were obtainedfrom Applied Biosystems GmbH (Weiterstadt,Germany).HCG β  oligonucleotides were developed basedon previous published data (1,9,10)  to amplify theHCG β  5, 8, 3, and 7 mRNAs, without amplifica-tion of HCG β   1, HCG β  2, or luteinizing hormone(LH)- β  mRNA. The primers yielded a RT-PCRproduct of 199 nucleotides. The distance betweenthe 3' end of the forward primer and the 5' end of the probe spans 18 bases. The optimal distancefor the 5'–3' nuclease activity of rTth DNA poly-merase seems to be below 50 bases. The univer-sal probe was placed close to the 5' end of exon 2.In cases of contaminating DNA amplification, thedifference between the 3' primer end and the 5'probe end is 371 bases and would lead to an in-sufficient 5'–3' nucleolytic activity of rTth poly-merase. The forward primer and probe have onlya 1 bp mismatch, and the reverse primer has a 2bp mismatch with the LH β  gene. Although thenucleotide sequences of HCG β  and LH β  are 94%homologous, we excluded an amplification of LH β   transcripts by DNA sequencing. 2.3. PCR Product Sequencing  Direct sequencing of the PCR products wasperformed with the automatic DNA sequencer ABI PRISM 310 Genetic Analyzer (PE AppliedBiosystems) using ABI dye-terminator chemis-try according to manufacturer’s protocol. The  Human Chorionic Gonadotropin-  β   mRNA49  M  OLECULAR   B  IOTECHNOLOGY  Volume 14, 2000  sequencing reactions were repeated at leasttwice in both directions from independent tem-plate preparations to avoid the possibility of PCR artifacts. 2.4. Preparation of RNA Standard  For calibration of the HCG β  TaqMan assay, aRNA standard was generated by using an in vitroT7-polymerase transcription system (Ribo-MAX™ Large Scale RNA Production System;Promega, Madison, WI). Using the TaqMan 5'and 3' primers, a preparative standard PCR reac-tion was performed to produce a HCG β -specificDNA fragment. The cloning procedure was car-ried out by ligating this fragment into a SmaI   lin-earized pBLUESCRIPT KS vector (Stratagene,La Jolla, CA).Several clones were analyzed by sequencing tovalidate the identity of the HCG β  fragment and tofind out the correct strand orientation in relationto the T7 promoter. The correct plasmid clone waspurified by ionic exchange column DNA prepa-ration (Qiagen, Valencia, CA) and linearized by  XhoI   cleavage to generate a useful template for producing a cut-off in vitro transcript. In vitrotranscription was done according to themanufacturer’s protocols. A DNase I digestionwas performed to remove the remaining DNAtemplate followed by phenol/chloroform extrac-tion and isopropanol precipitation. After photo-metric quantification of the newly synthesizedRNA, the absolute number of molecules per vol-ume was calculated.A starting mixture was made by diluting thespecific RNA molecules with a competitor yeasttRNA (Life Technologies) to a final concentra-tion of 10 10  in vitro transcripts in 2 µ g yeast tRNAper 10 µ L (in water). The addition of competitor RNA is intended to minimize unspecific surface-adhesion effects, as well as to compete for enzy-matic or chemical destruction of the specific mol-ecules. This starting mixture was used to preparestock dilution series over eight logs from 10 9  to10 2  HCG β   specific RNA molecules, each vialcomplemented by yeast tRNA to a final con-centration of 2 µ g/10 µ L. The stock dilution Fig. 1. Schematic representation of HCG β  gene (7–7665 bp). The three exons are indicated by boxes, theintrons by lines. The arrangement and nucleotide sequence of primers and TaqMan probe are shown.  50 Reimer et al. M  OLECULAR   B  IOTECHNOLOGY  Volume 14, 2000  series, always stored at –80 ° C, serves as a long-term source for a working dilution series madeby diluting aliquots 1 to 10 without supple-menting the working dilution with additionalcompetitor tRNA.This final procedure permits an easy pipetingof 10 µ L aliquots each containing 200 ng com-petitor tRNA, suitable for rTth DNA polymeraseTaqMan PCR, and eight different calibrationamounts of the specific RNA molecules extend-ing from 10 8  to 10 1 . 2.5. RT-PCR Procedure  The TaqMan EZ RT-PCR Kit (PE AppliedBiosystems) was used for reverse transcriptionand amplification of both targets and standards.Production of cDNA and PCR amplification wascarried out in a single-tube, single-enzyme sys-tem without the addition of subsequent enzymesor buffers. To obtain RT-PCR conditions with re-duced variability, PCR premixes containing allreagents except for targets were prepared andaliquoted into optical reaction tubes (PE AppliedBiosystems). No template controls (NTCs), stan-dards, and unknown samples were added in a vol-ume of 10 µ L. Placental tissue served as positivecontrol. Potential PCR product contaminationswere digested by uracil-  N  -glycosylase (UNG),since dTTP is substituted by dUTP. All RT-PCRreactions were performed in duplicate with a finalvolume of 50 µ L.RT-PCR conditions were optimized for primer,probe, template, and manganese acetate concen-trations (Table 1) . The reaction conditions were2 min at 50 ° C, 30 min at 60 ° C, 5 min at 95 ° C, 45cycles with 20 sec at 94 ° C, and 1 min at 60 ° C. 2.6. Quantification of HCG  β  mRNA Expression  PCR assays were performed in a 96-wellmicrotiter plate format on an ABI PRISM 7700Sequence Detector System (PE AppliedBiosystems). The first two lanes contained serialdilutions of the prepared standard with knownamounts of input RNA molecules to create a stan-dard curve. Each well was screened for fluores-cent dye every 7 sec and signals were regarded aspositive if the fluorescence intensity exceeded10X the standard deviation of the baseline fluo-rescence (threshold cycle,  C t  ). The C t   value of each well was compared to the standard curve of plasmid RNA. 2.7. Purification of Glycodelin-A Glycodelin-A was purified from human mid-trimester amniotic fluid according to the methoddescribed by Riittinen et al. (11)  with some modi-fications. Purification by use of a final HPLC stepresults in an electrophoretic pure product whenanalyzed by sodium dodecyl sulfate polyacryla-mide gel electrophoresis (SDS-PAGE). However,glycodelin-A prepared by this procedure did notstimulate cultured trophoblast cells to secrete syn-cytiotrophoblast markers such as HCG, humanplacental lactogen (HPL), or progesterone (datanot shown). Therefore, we modified the prepara-tion of glycodelin-A. Glycodelin-A was finallypurified on a Resource-Phe column (Pharmacia,Uppsala, Sweden) after hydrophobic interactionchromatography on octyl-Sepharose (Pharmacia).This purification method produced one 28 kDaband as shown by SDS-PAGE. N-terminal aminoacid sequence of MDIPQ for the first five aminoacids was obtained for the isolated protein. Thissequence is identical to that reported for humanglycodelin-A  (12) . 2.8. Culture of Trophoblasts  Cytotrophoblast cells were isolated from threedifferent human placentas after spontaneousdelivery or planned cesarean section as describedpreviously  (13) . Villous tissue dissected from theplacenta was minced and the decidua wasremoved. The remaining trophoblast tissue wasminced and transferred to HBSS-HEPES-buffer containing trypsin and DNase I. The cell suspen-sions obtained by digestion steps were separatedfrom tissue fragments. Trophoblast cell purifica-tion was performed on a Percoll gradient. Tropho-blast cells were removed, washed, and suspendedin RPMI 1640 culture medium supplemented with10% fetal calf serum (FCS) and gentamicin (200 µ g/mL).Trophoblast identity of the isolated cells wasdetermined morphologically by light microscopy,  Human Chorionic Gonadotropin-  β   mRNA51 M  OLECULAR   B  IOTECHNOLOGY  Volume 14, 2000  Table 1 Composition and Optimization Strategies for HCG β  RT-PCR AssayComponentOptimization stepsOptimalconcentration5X TaqMan EZ BufferNone10 µ LManganese acetate (25m  M  )2m  M  , 3m  M  , 4m  M  , 5m  M  3 m  M  dATP (10m  M  )None300 µ  M  dCTP (10m  M  )None300 µ  M  dGTP (10m  M  )None300 µ  M  dUTP (20m  M  )None600 µ  M  Forward primer (10 µ  M  )Primer matrix: 100n  M  , 300n  M  , 900n  M  300n  M  Reverse primer (10 µ  M  )Primer matrix: 100n  M  , 300n  M  , 900n  M  300n  M  TaqMan probe (5 µ  M  )100nM, 200n  M  100n  M  rTth DNA Polymerase (2.5 U/  µ L)None0.1 U/  µ LAmpErase UNG (1 U/  µ L)None0.01 U/  µ LRNA content in 10 µ L template100 ng, 200 ng, 400 ng, 600 ng, 800 ng, 1 µ g200 ngDEPC-H 2 Oad 50 µ L by their ability to produce specific hormone HCG,and by immunostaining with the anticytokeratinantibody AE1/AE3 (Boehringer Mannheim,Mannheim, Germany). 2.9. Treatment of Trophoblast Culture with Glycodelin-A Purified trophoblasts were adjusted to a cellconcentration of 1 × 10 6  cells/mL in RPMI 1640culture medium supplemented with 10% FCS andgentamicin (200 µ g/mL). The cell suspension (6mL) was incubated in cell culture flasks in hu-midified 5% CO 2  –95% air at 37 ° C in the pres-ence of 150 µ g/mL glycodelin-A. Untreated cellswere used as controls. 3. Results To determine the linear relationship betweenthe C t  and the log starting copy number, definedamounts of standard molecules of RNA dilutionswere amplified. As shown in Fig. 2 , quantifica-tion of HCG β  RNA standard was linear over eightlogs and the assay can measure as little as 100copies of HCG β  mRNA copies per tube. The C t  values decreased linearly with increasing targetquantity. All duplicate amplifications resulted invery similar C t  values. In all experiments the cor-relation coefficient was above 0.90.To exclude amplification of contaminatingDNA, we tested genomic first-trimester placentaDNA as a template for our described HCG β  probeand primers. Using various concentrations of genomic placental DNA (100–800 ng per run), noamplifications were observed in conventionalPCR (data not shown) or TaqMan assay (Fig. 3a) .To rule out amplification of related sequences, aLH β  RNA template was generated from a femaleperimenopausal hypophysis (provided by theDepartment of Pathology, University of Rostock).No RT-PCR products were seen for different hy-pophysial LH β  RNA concentrations when per-forming the established HCG β  TaqMan™ assay (Fig. 3b). We carried out RT-PCR on a large panel of to-tal RNAs extracted from normal tissues to mea-sure the absolute copy number of HCG β  mRNA.Quantitative results were categorized into four groups according to their expression of HCG β mRNA (Table 2) . High levels of HCG β  mRNAs(>1000 molecules per tube) were detected in pla-centa, uterus, and testis. Nontrophoblastic tissuesof seven distinct srcins (thymus, skeletal muscle,
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