Optimization of RNA extraction from formalin-fixed, paraffin-embedded lymphoid tissues.
Comprehensive analysis of gene expression using RNA extracted from frozen lymphoma specimens is becoming increasingly important for understanding disease pathogenesis, disease subclassification, and prognostication. As paraffin tissues are widely available whereas frozen specimens are not, development of gene expression analysis based on RNA extracted from paraffin-embedded tissues would facilitate application of the accumulated knowledge to a sample type that is typical of clinical practice. In the present study, we have developed and optimized methods of RNA extraction from paraffin-embedded lymphoid tissues. In contrast to previously suggested methods of RNA extraction from paraffin, our method uses sodium dodecyl sulfate that better preserves the extracted RNA and is optimized for more complete proteinase K digestion to release RNA from its complexes with protein. These modifications yield long RNA fragments up to 2000 bp enabling amplification of long amplicons. This allows usage of paraffin specimens for molecular rescue of RNA transcripted from rearranged clonal immunoglobulin genes-an advance that may increase the eligibility of lymphoma patients for immunotherapeutic approaches. Furthermore, real-time polymerase chain reaction analysis of expression of genes implicated in determination of prognosis of diffuse large B-cell lymphoma patients demonstrated an extremely high correlation (R>0.90) in normalized gene expression between paired frozen and formalin-fixed, paraffin-embedded specimens. Similarly, good correlation was also observed in gene array studies. These results suggest that the methods of RNA extraction we propose are suitable for giving accurate real-time quantitative reverse transcriptase-polymerase chain reaction results, array gene expression profiling, and molecular rescue of RNA transcripted from rearranged immunoglobulin genes for diagnostic and immunotherapeutic approaches.
Chen
Jun
J
Department of Medicine, Division of Hematology-Oncology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA [corrected]
Byrne
Gerald E
GE
Lossos
Izidore S
IS
D005404:Fixatives; D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics; D005803:Genes, Immunoglobulin / Q000235:genetics; D006801:Humans; D008198:Lymph Nodes / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D008206:Lymphatic Diseases / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008224:Lymphoma, Follicular / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016612:Paraffin Embedding / Q000379:methods; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005404:Fixatives; D005557:Formaldehyde; D012313:RNA