Comparison of real-time PCR and nested PCR for the detection of Y chromosome sequences in the peripheral blood mononuclear cells of patients with systemic sclerosis

M Mosca¹, T Giuliano¹, M Curcio², M Doveri¹, F De Feo¹, C Tani¹, L Bazzichi¹, S Bombardieri¹

¹ Rheumatology Unit, Department of Internal Medicine, University of Pisa, Italy
² Immunohematology and Transfusional Medicine Unit, Azienda Ospedaliera Universitaria Pisana, Pisa, Italy

Correspondence to:
M Mosca, Rheumatology Unit, Department of Internal Medicine, University of Pisa, Via Roma 67, 56126 Pisa, Italy; marta.mosca@int.med.unipi.it

Accepted 24 March 2008

The first 150 words of the full text of this article appear below.

Real-time PCR (RT-PCR) is a sensitive technique that has been recently applied to the determination and quantification of fetomaternal microchimerism.^1–3 In a previous manuscript, we detected the presence of Y chromosome sequences in female patients with systemic lupus erythaematosus (SLE) using nested PCR.⁴ In the present study we evaluated the presence of Y chromosome sequences in the peripheral blood mononuclear cells (PBMCs) of female patients with systemic sclerosis (SSc) using nested and RT-PCR, correlating the results with the clinical and serological manifestations of the patients.

Genomic DNA was isolated from EDTA anticoagulated peripheral blood under a biosafety hood to avoid contamination, using a QIAamp Blood Kit (Qiagen GmbH, Hilden, Germany) and following the manufacturer’s blood and body fluid protocol. DNA was extracted from patient PBMCs (200 µl sample per column), eluted in a final volume of 80 µl and stored at –20°C until used.

A specific Y-chromosome sequence was detected . . . [Full text of this article]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?