The PAXgene(®) tissue system preserves phosphoproteins in human tissue specimens and enables comprehensive protein biomarker research.
Author(s): Gündisch S, Schott C, Wolff C, Tran K, Beese C, Viertler C, Zatloukal K, Becker KF
Publication: PLoS One, 2013, Vol. 8, Page e60638
PubMed ID: 23555997 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 (PAXgene fixation, snap-frozen, or formalin fixation) on the extractability and analysis of phosphoproteins by Western blot, ELISA, and 2-dimentional SDS-PAGE. A total of 24 tissue specimens were used in this study including ovarian, breast, and prostate cancer specimens, a leiomyosarcoma, and non-malignant stomach, esophagus, colon, duodenum, uterus, ovary, prostate, bladder, breast, liver, spleen, gall bladder, pancreas, skin, muscle, tongue, salivary gland, lung, thyroid, and cystadenoma specimens. Each tissue was divided into equal parts and either snap-frozen in liquid nitrogen, fixed by PAXgene Tissue Fix, or fixed in formalin. PFPE specimens were transferred to PAXgene Stabilizer reagent and were then processed identically to FFPE specimens. Protein extraction was performed using the Qproteome FFPE Tissue Kit for all specimens, with modifications to the suggested protocol for PFPE and snap-frozen specimens, and different extraction buffers used for each ELISA kit.
Summary of Findings:
Protein from PFPE and cryopreserved specimens performed similarly in Western blot analysis using 13 antibodies, including 11 directed against phosphoproteins, and both types of samples showed stronger signals in both the malignant and non-malignant specimens than protein extracted from FFPE tissue. Two-dimensional gel electrophoresis was unsuccessful for FFPE specimens, but it was generally similarly successful for PFPE and cryopreserved specimens. All three ELISAs were successful using protein extracted from cryopreserved specimens, but only the Erk-1/2 and phosphor-Erk-1/2 ELISAs (specific for denatured proteins) were successful for PFPE specimens (FFPE specimens were not included in these experiments). Tissue-specific differences were observed in the signal intensities of the differently preserved specimens for all three ELISAs. Lower signal intensity was observed for PFPE samples than cryopreserved samples for stomach and uterus specimens, but not the duodenum specimens, in the Erk-1/2 ELISA, but these differences were not apparent in Western blot analysis, and all other observed differences were.
Biospecimens
- Tissue - Ovary
- Tissue - Breast
- Tissue - Prostate
- Tissue - Muscle (Skeletal)
- Tissue - Stomach
- Tissue - Esophagus
- Tissue - Colorectal
- Tissue - Small Bowel
- Tissue - Uterus
- Tissue - Bladder
- Tissue - Liver
- Tissue - Spleen
- Tissue - Gall Bladder
- Tissue - Pancreas
- Tissue - Skin
- Tissue - Tongue
- Tissue - Salivary Gland
- Tissue - Lung
- Tissue - Thyroid Gland
- Tissue - Other
Preservative Types
- Formalin
- Frozen
- PAXgene
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Carcinoma
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform Protein ELISA Protein Western blot Protein 1D/2D gels Protein Colorimetric assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
PAXgene
Snap frozen
Preaquisition Diagnosis/ patient condition Malignant
Non-malignant
Biospecimen Acquisition Biospecimen location Ovary
Breast
Prostate
Muscle
Stomach
Esophagus
Colon
Duodenum
Uterus
Bladder
Liver
Spleen
Gall bladder
Pancreas
Skin
Tongue
Salivary gland
Lung
Thyroid
Cystadenoma
Analyte Extraction and Purification Analyte isolation method Non-denaturing cell extraction buffer
Mild non-denaturing cell extraction buffer
Denaturing cell extraction buffer
ELISA Specific Targeted peptide/protein Non-denatured Akt/Proteinkinase B
Denatured Erk-1/2
Denatured phosphor-Erk-1/2
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Study Purpose
The purpose of this study was to determine the impacts of fixation time, stabilization time, storage temperature, and storage duration on protein yield and quality for PAXgene-fixed tissue specimens. A total of 18 malignant and non-malignant tissue specimens were used in this study including pancreas, colon, liver, stomach, spleen, muscle, kidney, duodenum, and breast specimens. Protein extraction was performed using the Qproteome FFPE Tissue Kit with modifications to the suggested protocol.
Summary of Findings:
PAXgene fixation times ranging from 24 h to 120 h did not significantly affect protein yield or quality, as evaluated by Western blot analysis. The authors report that fixation times as short as 4 h also had no impact on the yield or immunoreactivity of protein extracted from PFPE tissue. Likewise, leaving tissues in PAXgene Tissue Stabilizer for 24 h up to 96 h had no significant effects on extracted protein yield or quality. However, when comparing average yields from pancreas and colon specimens stabilized for 24 h versus >24 h, there was a slight decrease in protein yield for pancreas specimens (7%), but not the colon specimens, after stabilization for >24 h. Although 30% decreases in protein yields were observed for 2 out of 5 individual specimens stored for at least one year at room temperature compared to 4 degrees C, combined specimen averages showed no differences between storage temperatures. Further, tissue-specific phosphorylation patterns were not affected by storage temperature. Storage of PFPE tissues at 4 degrees C for up to 18 months resulted in an average decrease in the yield of extracted proteins of 9% compared to that extracted after 6 months of storage. Western blot analysis was not affected by the additional year of 4 degrees C storage.
Biospecimens
- Tissue - Pancreas
- Tissue - Colorectal
- Tissue - Liver
- Tissue - Stomach
- Tissue - Breast
- Tissue - Muscle (Skeletal)
- Tissue - Kidney
- Tissue - Small Bowel
- Tissue - Spleen
Preservative Types
- PAXgene
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Carcinoma
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform Protein Colorimetric assay Protein Western blot Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 4 h
24 h
48 h
72 h
120 h
Storage Short-term storage solution 24 h in PAXgene Stabilizer
48 h in PAXgene Stabilizer
72 h in PAXgene Stabilizer
96 h in PAXgene Stabilizer
Storage Storage temperature 4 degrees C
Room temperature
Storage Storage duration 6 months
18 months
Preaquisition Diagnosis/ patient condition Malignant
Non-malignant
Biospecimen Acquisition Biospecimen location Pancreas
Colon
Liver
Stomach
Spleen
Muscle
Kidney
Duodenum
Breast
Western blot Specific Targeted peptide/protein Phosphorylated tyrosine residues
Beta-actin