DNA and RNA were isolated from clinical samples and Nthy-ori3-1 cells as described [27] (link). All RNA preparations were devoid of DNA, as assured by multiple DNase digestions and lack of amplification in PCR reactions where retrotranscription (RT) had been omitted [14] (link). HHV-6 DNA presence and load were analyzed by PCR and real time quantitative (qPCR) specific for the U94 and U42 genes [27] (link), and samples were considered positive when 1 µg of cell DNA harbored more than 100 copies of viral DNA [27] (link). Amplification of the house-keeping human RNase P gene was used as a control. All clinical samples were analyzed in a randomized and blinded fashion. In addition, 15/28 control and 21/34 HT FNAs, when there was enough material to repeat the analysis, were tested again in a randomized and blinded fashion at a distant time from the first analyses.
HHV-6 variant A or B identification was obtained by restriction enzyme digestion with HindIII enzyme of the U31 nested PCR amplification product, as reported previously [26] (link). Digestion products were then visualized on ethidium bromide stained agarose gel after electrophoresis migration.
Virus transcription was assessed by PCR or qPCR after retrotranscription (RT-PCR, RT-qPCR), determining the presence of lytic (U42, U22) or latent (U94 in the absence of U42) mRNAs, as previously reported [27] (link). The sensitivity of the used PCRs was similar for all genes, detecting as few as 100 copies of target sequence.
Cell fractions derived by immunomagnetic separation of FNAs were characterized by RT-PCR specific for leukocytes transcripts (respectively CD45, CD3), using serial dilutions of cDNA template, corresponding to amounts of total extracted RNA ranging from 100 ng to 1 pg. Primers and PCR conditions for CD3 and CD45 were previously reported [53] (link), [54] (link), and amplification reactions were carried out for 30 cycles. In each assay the cDNAs obtained from JJhan T cells or Nthy-ori3-1 thyroid cells were also included as positive and negative controls respectively. Amplification of the house-keeping β-actin gene was used as a control.
HHV-6 variant A or B identification was obtained by restriction enzyme digestion with HindIII enzyme of the U31 nested PCR amplification product, as reported previously [26] (link). Digestion products were then visualized on ethidium bromide stained agarose gel after electrophoresis migration.
Virus transcription was assessed by PCR or qPCR after retrotranscription (RT-PCR, RT-qPCR), determining the presence of lytic (U42, U22) or latent (U94 in the absence of U42) mRNAs, as previously reported [27] (link). The sensitivity of the used PCRs was similar for all genes, detecting as few as 100 copies of target sequence.
Cell fractions derived by immunomagnetic separation of FNAs were characterized by RT-PCR specific for leukocytes transcripts (respectively CD45, CD3), using serial dilutions of cDNA template, corresponding to amounts of total extracted RNA ranging from 100 ng to 1 pg. Primers and PCR conditions for CD3 and CD45 were previously reported [53] (link), [54] (link), and amplification reactions were carried out for 30 cycles. In each assay the cDNAs obtained from JJhan T cells or Nthy-ori3-1 thyroid cells were also included as positive and negative controls respectively. Amplification of the house-keeping β-actin gene was used as a control.
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