title: A simple and efficient method for DNA extraction from skin and paraffin-embedded tissues applicable to T-cell clonality assays.
abstract: PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.
authors: Department of Molecular Hematology, National Hematology Research Center, Moscow, Russia.
authors: Julia V
authors: JV
authors: Sidorova
authors: Bella V
authors: BV
authors: Biderman
authors: Elena E
authors: EE
authors: Nikulina
authors: Andrey B
authors: AB
authors: Sudarikov
mesh_terms: D019342:Acetic Acid / Q000737:chemistry; D000641:Ammonia / Q000737:chemistry; D004247:DNA / Q000302:isolation & purification*; D015329:Gene Rearrangement, T-Lymphocyte; D006801:Humans; D016612:Paraffin Embedding; D012867:Skin / Q000737:chemistry*
chemical_list: D000641:Ammonia; D004247:DNA; D019342:Acetic Acid