A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

Author(s): Chu Wei-Sing, Liang Qi, Liu Jilan, Wei Min Q, Winters Mary, Liotta Lance, Sandberg Glenn, Gong Maokai

Publication: Lab Invest, 2005, Vol. 85, Page 1416

PubMed ID: 16127423 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   To access the yield and size of nucleic acids and proteins extracted from FFPE or frozen tissue section using the NDME method. Tissues examined include brain, breast, heart, pancreas, liver, lung, spleen, lymph node, colon, and prostate.

   Summary of Findings:

   As determined by coomassie blue and silver staining, protein and nucleic acid yields were significantly greater compared to other extraction buffers, and included high molecular weight species. Nucleic acid yield was reduced (75%) in extracts from FFPE tissue sections compared to frozen sections.

   Biospecimens

   • Tissue - Lymph Node
   • Tissue - Breast
   • Tissue - Heart
   • Tissue - Brain
   • Tissue - Pancreas
   • Tissue - Lung
   • Tissue - Liver
   • Tissue - Colorectal
   • Tissue - Spleen
   • Tissue - Prostate

   Preservative Types

   • Formalin
   • OCT

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels
   RNA	Electrophoresis
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) OCT
   Analyte Extraction and Purification	Analyte isolation method	NMDE-PE FFPE-NE 0.01 M EDTA 10 mM citrate Tissue-PE LB

   View more

2. Study Purpose

   To determine if cellular structure is compromised after NDME treatment of tissue sections.

   Summary of Findings:

   Cellular structure remained preserved after NDME extraction. Further, hematoxylin and eosin staining was enhanced after NDME treatment, with more vivid staining, reduction of intercellular spaces, and slight swelling of nucleoli.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Morphology	H-and-E microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)

   View more

3. Study Purpose

   To assess the effects of NDME treatment on CD5 antigenicity as determined by immunohistochemistry.

   Summary of Findings:

   CD5 immunopositive staining revealed that antigen retrieval was not necessary in NDME treated tissue sections. Both the intensity of the immunohistochemical signal and the protein yield in the extraction buffer increased with the length of extraction, plateauing at 30 min, however, incubations longer than 20 min compromised structural integrity of the tissue section.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Incubation duration/condition	0 min 5 min 10 min 15 min 20 min 30 min

   View more

4. Study Purpose

   To determine if protein size and antigenicity are compromised after NDME.

   Summary of Findings:

   Western blot analysis for representative membrane glycoproteins, HIV capsid, Golgi precursor, and nuclear proteins displayed appropriate bands at the predicted sizes. Futher, use of NDME-samples appears to increase platform sensitivity, with a single tissue section extract able to generate a detectable Western blot signal. Microarray analysis of NDME treated protein extracts revelaed expression of common proteins in the majority of tissues examined, as well as appropriate expression of tissue-specific proteins.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin
   • Ethanol
   • Other Preservative
   • OCT

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Western blot
   Protein	Reverse phase protein microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	NDME Ethanol Formalin (buffered) OCT

   View more

5. Study Purpose

   To determine if protein profiles are compromised after NDME.

   Summary of Findings:

   Extracts from frozen and FFPE sections (enriched and desalted) exhibited similar protein profiles, within low and high (10-21K m/z) mass/charge (m/z) ranges.

   Biospecimens

   • Tissue - Pancreas

   Preservative Types

   • Formalin
   • OCT

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	SELDI-TOF MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	NDME
   Biospecimen Preservation	Type of fixation/preservation	OCT Formalin (buffered)

   View more

6. Study Purpose

   To determine if the integrity of NDME nucleic acids is sufficient for molecular analysis.

   Summary of Findings:

   Successful PCR analysis of DNA and RNA extracted using the NDME method indicated nucleic acid integrity was preserved (amplicon size range: 354-1309 bp). However, mRNA quantity was significantly lower (50%) in FFPE sections compared to frozen tissue sections. In situ hybridization post NDME yielded appropriate signal localization and preservation of cellular detail.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • OCT

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	In situ hybridization
   RNA	In situ hybridization
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) OCT

   View more