%0 Journal Article
%T Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi's sarcoma
%A Vivian Kour¨ª
%A Pedro A Mart¨ªnez
%A Orestes Blanco
%A Virginia Cap¨®
%A Mar¨ªa E Rodr¨ªguez
%A Mar¨ªa del C Dovigny
%A Lidia Cardell¨¢
%A Angela Gala
%A Narciso A Jim¨¦nez
%A Consuelo Correa
%A Yoan Alem¨¢n
%A Lissette P¨¦rez
%A Alina ¨¢lvarez
%A Ulrich Hengge
%J Herpesviridae
%D 2010
%I BioMed Central
%R 10.1186/2042-4280-1-3
%X One hundred and forty-two clinical samples belonging to 49 patients with epidemic KS histologically diagnosed in the Pathology Department at the Institute of Tropical Medicine Pedro Kour¨ª between 2004-2007 were included. The research was approved by local and national ethics committees; all subjects provided their written informed consent. Clinical, immunological and epidemiological data from each patient are depicted in Table 1.Different types of samples (41 saliva, 48 tissues, 26 PBMC, and 27 plasma) obtained from each individual were tested at the same time. PBMC and plasma were obtained by separation of 20 mL of citrated whole blood using a Ficoll separation gradient (SIGMA, UK). Paraffin was removed from tissues by xylene treatment according to published protocols [1]. DNA extraction was performed using the QIAamp£¿ DNA Mini Kit (QIAGEN, Germany) and the genomic DNA (gDNA) concentration was determined using spectrophotometer (GeneQuant II, Pharmacia Biotech, EUA) and adjusted to 100 ng, with the exception of plasma where 10 uL were directly used since it was not possible to quantify the gDNA. In order to obtain the standard DNA for absolute quantification, gDNA was extracted from the BCBL-1 cell line and the DNA copy numbers was determined by spectrophotometer as well. Then, the OD concentration was converted to DNA copy number following methods published elsewhere [2]. Once the gDNA copy number from BCBL-1 cell line was calculated, it was adjusted to 1 million copies and a standard curve with serial ten-fold dilutions (from 106 to 10 copies) assayed in triplicates was prepared. Once the run finished, the standard curve was automatically generated by the LightCycler software version 3.3, using the Second Derivative Maximum (SDMM) method. In additon, the detection of Human ¦Â globin gene was used as internal control in each clinical sample (with exception of plasma) for measuring of the exact amount of input DNA [3].RT-PCR primers and conditions were described pre
%U http://www.herpesviridae.org/content/1/1/3