Proteomic analysis of PAXgene-fixed tissues.
Author(s): Ergin B, Meding S, Langer R, Kap M, Viertler C, Schott C, Ferch U, Riegman P, Zatloukal K, Walch A, Becker KF
Publication: J Proteome Res, 2010, Vol. 9, Page 5188-96
PubMed ID: 20812734 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of preservation method (cryopreserved, PAXgene fixation, or formalin fixation) and duration of PAXgene fixation on protein yield and expression as determined by 1D gels, Western blot analysis, reverse phase protein microarrays, and MALDI-TOF MS analysis of an unspecified number of nonmalignant spleen, breast, duodenum, kidney, pancreas, and stomach specimens. Protein was extracted using the QProteome FFPE Tissue-Kit for all specimens, with a modified protocol for snap frozen and PFPE specimens.
Summary of Findings:
Ponceau S staining revealed comparable protein patterns after optimization of extraction protocols for PFPE, cryopreserved, and FFPE tissues. Western blot analysis showed less intense bands for FFPE tissue than PFPE or cryopreserved specimens, while the latter two were comparable for most antibodies. In most tissues, there were no differences in Western analysis results between specimens fixed in PAXgene for 3 versus 24 h, however, weaker band intensities were apparent in breast tissue fixed for 24 h rather than 3 h for. Protein extracted from all specimens showed clear spots using extracellular signal-regulated kinase (ERK) antibody for reverse phase protein microarray analysis, but the authors mention that protein extracted from FFPE tissue showed stronger spot intensities than protein from PFPE or cryopreserved specimens. MALDI-TOF MS analysis of samples extracted from FFPE tissue only detected 2 peaks in the endocrine pancreas and none for the exocrine pancreas but successfully detected over 100 peaks for protein extracted from PFPE and cryopreserved specimens. MALDI-TOF Signal intensities for insulin and glucagon were higher for cryopreserved specimens than PFPE specimens.
Biospecimens
- Tissue - Spleen
- Tissue - Breast
- Tissue - Stomach
- Tissue - Kidney
- Tissue - Pancreas
- Tissue - Small Bowel
Preservative Types
- Formalin
- Frozen
- PAXgene
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform Protein Colorimetric assay Protein Western blot Protein Reverse phase protein microarray Protein MALDI-TOF MS Protein 1D/2D gels Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Spleen
Breast
Duodenum
Kidney
Pancreas
Stomach
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
PAXgene
Biospecimen Preservation Time in fixative 3 h
24 h
Western blot Specific Targeted peptide/protein E-cadherin
Beta actin
ERK
phosphorylated ERK
Hsp70
Reverse phase protein microarray Specific Targeted peptide/protein ERK
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Study Purpose
The purpose of this study was to determine the effects of preservation method (snap-frozen, PAXgene fixation, or formalin fixation) and duration of PAXgene fixation on RNA yield, integrity, and RT-PCR analysis of an unspecified number of nonmalignant liver specimens. RNA was extracted from PFPE tissue using the PAXgene Tissue RNA Kit, from FFPE tissue using the RNeasy FFPE Kit, and from snap-frozen specimens using Trizol.
Summary of Findings:
RT-PCR amplification of 3 different fragments of GAPDH was successful for cryopreserved and PFPE specimens, regardless of time in fixative, but the largest fragment (530 bp) was not successfully amplified from FFPE specimens. Electrophoresis of total RNA showed intact 28S and 18S rRNA for cryopreserved and PFPE specimens but not for FFPE specimens. Calculated RINs for the differently preserved specimens were 8.5 (cryopreserved), 5.0 (PFPE 3 h), 5.3 (PFPE 24 h), and 2.3 (FFPE).
Biospecimens
Preservative Types
- Formalin
- Frozen
- PAXgene
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform RNA Electrophoresis RNA RT-PCR RNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
PAXgene
Snap frozen
Biospecimen Preservation Time in fixative 3 h
24 h
RT-PCR Specific Targeted nucleic acid GAPDH
RT-PCR Specific Length of gene fragment 153 bp
323 bp
530 bp
Analyte Extraction and Purification Analyte isolation method PAXgene Tissue RNA Kit
RNeasy FFPE Kit
Trizol