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

The purpose of this paper was to determine the effects of preservation method (cryopreserved, PAXgene fixation, or formalin fixation) and duration of PAXgene fixation on protein and RNA yield, integrity, and expression in nonmalignant tissue specimens.

Conclusion of Paper

Western blot analysis showed less intense bands for formalin-fixed paraffin-embedded (FFPE) tissue than PAXgene-fixed paraffin-embedded (PFPE) or cryopreserved specimens, while the latter two were comparable for most antibodies. Protein extracted from all specimens showed clear spots for reverse phase protein microarray analysis, but FFPE tissue showed stronger spot intensities than PFPE or cryopreserved specimens. MALDI-TOF mass spectrometry (MS) was unsuccessful (only 2 peaks) for the samples extracted from FFPE tissue but was successful (>100 peaks) for protein extracted from PFPE and cryopreserved specimens. Signal intensities for chosen targets insulin and glucagon were higher for cryopreserved specimens than PFPE specimens. RT-PCR amplification of 3 different fragments of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was successful for cryopreserved and PFPE specimens, regardless of time in fixative, but the largest fragment was not successfully amplified from FFPE specimens. RNA integrity numbers (RINs) were 8.5 (cryopreserved), 5.0 (PFPE 3 h), 5.3 (PFPE 24 h), and 2.3 (FFPE).

Studies

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

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

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

Tissue - Liver

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

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