A new technology for stabilization of biomolecules in tissues for combined histological and molecular analyses.
Author(s): Viertler C, Groelz D, Gündisch S, Kashofer K, Reischauer B, Riegman PH, Winther R, Wyrich R, Becker KF, Oelmüller U, Zatloukal K
Publication: J Mol Diagn, 2012, Vol. 14, Page 458-66
PubMed ID: 22749745 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of preservation method (snap frozen, PAXgene fixation, or formalin fixation) on protein, DNA, and RNA analyses using a variety of malignant and nonmalignant tissue specimens.
Conclusion of Paper
In general, analysis of protein, DNA, and RNA using PAXgene-fixed paraffin-embedded (PFPE) tissue was superior to using formalin-fixed paraffin-embedded (FFPE) tissue and comparable to the analysis of snap frozen specimens. Notable exceptions were that morphology was less well preserved in snap frozen specimens than PFPE and FFPE specimens, and RNA integrity numbers (RINs) were lower for RNA extracted from PFPE specimens than from snap frozen specimens.
Studies
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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) on morphological preservation and protein analyses using a variety of malignant, nonmalignant, and non-neoplastic tissue specimens. Protein was extracted from PFPE and snap frozen specimens by the QProteome FFPE Tissue Kit with protocol modifications and from FFPE specimens using the same kit's manufacturer suggested protocol.
Summary of Findings:
H&E staining revealed well preserved morphology in PFPE and FFPE specimens, while chromatin structure and other details were less well preserved in snap frozen specimens. Among MutL homolog 1 (MLH1) positive colon specimens, MLH1 immunostaining was similar between differently preserved specimens, although the authors state that less harsh antigen retrieval methods were necessary for PFPE specimens than for FFPE specimens (data not shown). Western blot analysis of full length proteins, including phosphorylated proteins, extracted from liver specimens was possible, regardless of preservation method. Slightly weaker band intensity was seen for some targets from FFPE tissue compared to snap frozen or PFPE tissue, but there were no noted differences between PFPE tissues fixed for 3 h versus 24 h.
Biospecimens
Preservative Types
- Formalin
- Frozen
- PAXgene
Diagnoses:
- Not specified
- Neoplastic - Benign
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Protein Western blot Morphology H-and-E microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
PAXgene
Snap frozen
Immunohistochemistry Specific Targeted peptide/protein MLH1
Western blot Specific Targeted peptide/protein p-NFkB
NFkB
p-Akt
Akt
p-GSK3Beta
GSK3Beta
p-PTEN
PTEN
cytokeratin 18
vimentin
GAPDH
Beta actin
Biospecimen Preservation Time in fixative 3 h
24 h
-
Study Purpose
The purpose of this study was to determine the effects of preservation method (snap frozen, PAXgene fixation, or formalin fixation) on RNA and microRNA (miRNA) analysis using a variety of malignant and nonmalignant tissue specimens. mRNA was extracted from 10-20 sections each of an unspecified number of specimens using the PAXgene Tissue RNA Kit for PFPE specimens, the RNeasy FFPE Kit for FFPE specimens, and the Invitrogen TRIzol procedure for snap frozen specimens. miRNA was extracted from three colon cancer specimens using PAXgene Tissue miRNA Kit for PFPE tissue, Recover All Total Nucleic Acid Isolation Kit for FFPE tissue, and the miRNeasy Mini Kit using TissueLyser II system for snap frozen specimens.
Summary of Findings:
RINs were 2.20 for FFPE tissue, 4.00 and 4.20 for PFPE tissue fixed for 24 and 3 h, respectively, and 8.00 for snap frozen specimens. Cycle threshold (Ct) values were similar between PFPE and snap frozen specimens, regardless of amplicon size, however, Ct values were higher for FFPE specimens for all amplicon lengths. A range in PAXgene fixation times between 3 and 120 h did not adversely affect mRNA quality or real time qRT-PCR results. A very strong correlation (r2=0.99) was observed between PFPE tissue and snap-frozen tissue for expression analysis of 92 cancer pathway genes, while the correlation was not as high between FFPE and snap frozen tissue (r2=0.89). Very strong (r2=0.95) and strong (r2=0.81) correlations were observed between snap frozen miRNA expression and PFPE or FFPE miRNA expression, respectively.
Biospecimens
- Tissue - Muscle (Skeletal)
- Tissue - Liver
- Tissue - Colorectal
- Tissue - Breast
- Tissue - Adipose
- Tissue - Kidney
- Tissue - Small Bowel
- Tissue - Spleen
- Tissue - Stomach
Preservative Types
- Formalin
- Frozen
- PAXgene
Diagnoses:
- Not specified
- Neoplastic - Benign
- Neoplastic - Carcinoma
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
PAXgene
Snap frozen
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
Real-time qRT-PCR Specific Length of gene fragment 71 bp
153 bp
200 bp
277 bp
323 bp
530 bp
Biospecimen Preservation Time in fixative 3 h
24 h
120 h
-
Study Purpose
The purpose of this study was to determine the effects of preservation method (snap frozen, PAXgene fixation, or formalin fixation) on DNA analysis using five colorectal cancer tissue specimens. DNA extraction was performed using PAXgene Tissue DNA Kit for PFPE tissue, QIAamp DNA FFPE Tissue Kit for FFPE tissue, and QIAamp DNA Mini Kit with TissueLyser II system for snap frozen specimens.
Summary of Findings:
Long-range and multiplex PCR, Sanger sequencing (data not shown), and pyrosequencing were all successful using DNA extracted from PFPE or snap frozen specimens. In contrast, DNA extracted from FFPE specimens was not successfully analyzed by these platforms.
Biospecimens
Preservative Types
- Formalin
- Frozen
- PAXgene
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Electrophoresis DNA PCR DNA DNA sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
PAXgene
Snap frozen
PCR Specific Technology platform Long range PCR
Multiplex PCR
PCR Specific Targeted nucleic acid Human tuberous sclerosis complex
Prion protein
Immunoglobulin-associated Beta protein
Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
Angiotensin II receptor type 2
CD14
CD40
CD59
CD19