Preparation of Chorionic Villus Samples for Metaphase Chromosome Analysis and Chromosomal Microarray Analysis

Amy Breman1, Ankita Patel1

1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.3
DOI:  10.1002/0471142905.hg0803s75
Online Posting Date:  October, 2012
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Abstract

Chorionic villi are composed of an outer layer of trophoblastic cells and an inner mesenchymal cell core. They can be prepared for chromosome analysis using a culture method wherein villi are disaggregated by mechanical and enzymatic methods and the resulting cell suspension is used to establish primary cultures. Mesenchymal cells of the villus core are released by this procedure and the fibroblasts are actively proliferative in tissue culture. Cultures can be used for cytogenetic analysis after ∼1 week. Slides prepared by this technique can be stained using trypsin‐Giemsa banding and analyzed for chromosomal abnormalities in fetal tissue. Chorionic villi may also be assessed by chromosomal microarray analysis (CMA). For this purpose, a method for extraction of high‐quality DNA from CVS tissue is also described here. Curr. Protoc. Hum. Genet. 75:8.3.1‐8.3.9. © 2012 by John Wiley & Sons, Inc.

Keywords: chorionic villi CVS; cytogenetics; metaphase; prenatal

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Metaphase Spreads from Chorionic Villi Using the Culture Method
  • Basic Protocol 2: Preparation of DNA from Chorionic Villi for Chromosomal Microarray Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Metaphase Spreads from Chorionic Villi Using the Culture Method

  Materials
  • Chorionic villus sample
  • Complete medium: AmcelGrow (ID Labs) or Amniomax (GIBCO)
  • 0.25% trypsin/EDTA solution (see recipe)
  • MEM CellGro with L‐glutamine (Mediatech)
  • Collagenase solution (see recipe)
  • 10 µg/ml colcemid (e.g., GIBCO)
  • 1% (w/v) sodium citrate, 37°C
  • Fixative: 3:1 (v/v) methanol/acetic acid, 4°C, freshly prepared
  • 35‐ and 60‐mm plastic Petri dishes
  • Watchmaker's fine forceps, sterile
  • Inverted microscope or dissecting stereomicroscope
  • Pasteur pipet, sterile
  • Scalpels with #10 blade (sterile, disposable)
  • 1‐, 5‐, and 10‐ml serological pipets, sterile
  • 15 ml polystyrene centrifuge tubes, sterile
  • Sorvall GLC‐2B centrifuge equipped with HL‐4 rotor and six 15‐ml swinging buckets (or equivalent)
  • 22 × 22–mm glass coverslips, sterilized
  • 35‐mm plastic Petri dishes
  • Vacuum pump for aspirating
  • Thermotron environmental chamber
  • 90°C oven (VWR 1330FM or equivalent)

Basic Protocol 2: Preparation of DNA from Chorionic Villi for Chromosomal Microarray Analysis

  Materials
  • Cleaned chorionic villus sample
  • 1× phosphate‐buffered saline (PBS, see recipe in appendix 2D or purchase, e.g., from Cambrex)
  • Cell Lysis Buffer (Puregene)
  • 20 mg/ml proteinase K (Qiagen)
  • 100 mg/ml RNase A (Qiagen)
  • 100% ethanol
  • QIAamp DNA Blood Midi Kit (Qiagen) with AW1 and AW2 wash buffers and Buffer AE
  • DNA Clean Concentrator‐5 kit (Zymo Research)
  • Molecular‐grade water (VWR)
  • Microcentrifuge (e.g., Eppendorf 5415D)
  • Rocking platform incubator (e.g., Boekel)
  • 15‐ml polystyrene centrifuge tubes, sterile
  • Benchtop centrifuge with swinging bucket rotor (e.g., Eppendorf 5810‐R)
  • 1.7‐ml microcentrifuge tubes (VWR)
  • NanoDrop ND‐1000 Spectrophotometer (NanoDrop)
  • Barrier pipet tips (VWR)
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Figures

Videos

Literature Cited

Literature Cited
   Brambati, B. and Oldrini, A. 1986. Methods of chorionic villus sampling. In Chorionic Villus Sampling (B. Brambati, G. Simoni, and S. Fabro, eds.) pp. 73‐98. Marcel Dekker, New York.
   Brambati, B., Oldrini, A., Ferrazzi, J., and Lanzani, A. 1987. Chorionic villus sampling: An analysis of the obstetric experience of 1000 cases. Prenatal Diagn. 7:157‐169.
   Hahnemann, N. 1974. Early prenatal diagnosis: A study of biopsy techniques and cell culturing from extraembryonic membranes. Clin. Genet. 6:294‐306.
   Hahnemann, N. and Vejerslev, L.O. 1997. Accuracy of cytogenetic findings on chorionic villus sampling (CVS) – diagnostic consequences of mosaicism and non‐mosaic discrepancy in centres contributing to EUCROMIC 1986‐1992. Prenat. Diagn. 17:801‐820.
   Jackson, L.G. 1986. Prenatal genetic diagnosis by chorionic villus sampling. In Perinatal Genetics: Diagnosis and Treatment (I.H. Porter, N.H. Hatcher, and A.M. Willey, eds.) pp. 95‐113. Academic Press, San Diego.
   Jackson, L.G. and Wapner, R.J. 1987. Risks of chorion villus sampling. Ballier's Clin. Obstet. Gynecol. 1:513‐532.
   Kazy, Z., Bakharev, V.A., and Stygar, A.M. 1979. Value of the ultrasonic studies in biopsy of the chorion, according to genetic indicators. Akush. Ginekol. 8:29‐31.
   Kazy, Z., Stygar, A.M., and Bakharev, V.A. 1980. Chorionic biopsy under immediate realtime (ultrasonic) control. Orv. Hetil. 121:2765‐2766.
   Kazy, Z., Rozovsky, I.S., and Bakharev, V.A. 1982. Chorionic biopsy in early pregnancy: A method of early prenatal diagnosis for inherited disorders. Prenatal Diagn. 2:39‐45.
   Mohr, J. 1968. Foetal genetic diagnosis: Development of techniques for early sampling of foetal cells. Acta Pathol. Microbiol. Immunol. Scand. 73:73‐77.
   Ou, Z., Kang, S.H., Shaw, C.A., Carmack, C.E., White, L.D., Patel, A., Beaudet, A.L., Cheung, S.W., and Chinault, A.C. 2008. Bacterial artificial chromosome‐emulation oligonucleotide arrays for targeted clinical array‐comparative genomic hybridization analyses. Genet. Med. 10:278‐289
   Sahoo, T., Cheung, S.W., Ward, P., Darilek, S., Patel, A., del Gaudio, D., Kang, S.H., Lalani, S.R., Li, J., McAdoo, S., Burke, A., Shaw, C.A., Stankiewicz, P., Chinault, A.C., Van den Veyver, I.B., Roa, B.B., Beaudet, A.L., Eng, C.M. 2006. Prenatal diagnosis of chromosomal abnormalities using array‐based comparative genomic hybridization. Genet. Med. 8:719‐727.
   Simoni, G., Brambati, B., Danesino, C., Rossela, F., Terzoli, G.L., Ferrari, M., and Fraccaro, M. 1983. Efficient direct chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy. Hum. Genet. 63:349‐357.
   Ward, R.H.T., Modell, B., Petrou, M., Karagozlu, F., and Douratsos, E. 1983. A method of chorionic villus sampling in the first trimester of pregnancy under real time ultrasonic guidance. Br. Med. J. 286:1542‐1544.
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