Optimization of RNA extraction from formalin-fixed, paraffin-embedded lymphoid tissues

Jun Chen, Gerald E. Byrne, Izidore Lossos

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)61-72
Number of pages12
JournalDiagnostic Molecular Pathology
Volume16
Issue number2
DOIs
StatePublished - Jun 1 2007

Fingerprint

Lymphoid Tissue
Paraffin
Formaldehyde
RNA
Gene Expression
Immunoglobulin Genes
Lymphoma
Endopeptidase K
Lymphoma, Large B-Cell, Diffuse
Gene Expression Profiling
Reverse Transcriptase Polymerase Chain Reaction
Sodium Dodecyl Sulfate
Real-Time Polymerase Chain Reaction
Digestion

Keywords

  • Formalin fixation
  • Immunoglobulin
  • Lymphoma
  • Paraffin-embedded tissue
  • RNA extraction

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Optimization of RNA extraction from formalin-fixed, paraffin-embedded lymphoid tissues. / Chen, Jun; Byrne, Gerald E.; Lossos, Izidore.

In: Diagnostic Molecular Pathology, Vol. 16, No. 2, 01.06.2007, p. 61-72.

Research output: Contribution to journalArticle

@article{b311e4270d56433aa53ae444891ae677,
title = "Optimization of RNA extraction from formalin-fixed, paraffin-embedded lymphoid tissues",
abstract = "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.",
keywords = "Formalin fixation, Immunoglobulin, Lymphoma, Paraffin-embedded tissue, RNA extraction",
author = "Jun Chen and Byrne, {Gerald E.} and Izidore Lossos",
year = "2007",
month = "6",
day = "1",
doi = "10.1097/PDM.0b013e31802f0804",
language = "English",
volume = "16",
pages = "61--72",
journal = "Diagnostic Molecular Pathology",
issn = "1052-9551",
publisher = "Lippincott Williams and Wilkins",
number = "2",

}

TY - JOUR

T1 - Optimization of RNA extraction from formalin-fixed, paraffin-embedded lymphoid tissues

AU - Chen, Jun

AU - Byrne, Gerald E.

AU - Lossos, Izidore

PY - 2007/6/1

Y1 - 2007/6/1

N2 - 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.

AB - 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.

KW - Formalin fixation

KW - Immunoglobulin

KW - Lymphoma

KW - Paraffin-embedded tissue

KW - RNA extraction

UR - http://www.scopus.com/inward/record.url?scp=34347372892&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34347372892&partnerID=8YFLogxK

U2 - 10.1097/PDM.0b013e31802f0804

DO - 10.1097/PDM.0b013e31802f0804

M3 - Article

C2 - 17525674

AN - SCOPUS:34347372892

VL - 16

SP - 61

EP - 72

JO - Diagnostic Molecular Pathology

JF - Diagnostic Molecular Pathology

SN - 1052-9551

IS - 2

ER -