Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for rt-PCR and microarray analysis.
Author(s): Kashofer K, Viertler C, Pichler M, Zatloukal K
Publication: PLoS One, 2013, Vol. 8, Page e70714
PubMed ID: 23936242 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
This study compared qRT-PCR performance in four different tissue types preserved by FFPE, TFPE, or by snap-freezing in liquid nitrogen-cooled isopentane. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplicons of different sizes (71-530 bp) were assessed in 99 samples collected from 28 patients by raw Ct value and gel electrophoresis of the end product. RIN was also evaluated as a predictor of qRT-PCR performance in snap frozen and FFPE liver specimens. RNA was extracted from snap frozen specimens with Trizol and from FFPE specimens using a modified phenol/chloroform method that had previously been reported.
Summary of Findings:
Amplification of all GAPDH fragments was successful using RNA isolated from snap-frozen thyroid, kidney, and duodenum specimens. For RNA from FFPE and TFPE specimens qRT-PCR success was influenced by tissue type and fragment size. Amplification of GAPDH was successful using RNA from FFPE kidney, thyroid and duodenum specimens when the GAPDH fragments were less than or equal to 150 bp, 200 bp, and 277 bp, respectively. Amplification was successful for all sized fragments of GAPDH using RNA from TFPE kidney and of GAPDH fragments less than or equal to 323 bp using RNA from TFPE thyroid and duodenum. When raw Ct values were plotted against amplicon size, the slope of the regression line was greater for RNA from FFPE specimens (2.4) compared to TFPE (0.55) or snap frozen (0.34) liver specimens. Raw Ct values also differed significantly when RNA from FFPE and snap frozen liver specimens were compared using a different specimen set (p<0.01). For RNA from snap frozen liver specimens, raw Ct values were modestly correlated to RIN (r= -0.05 to -0.6) while the correlation was weak (r= -0.25 to -0.28) for FFPE specimens. The authors also compared the efficacy of reverse transcription for each fixation method. For snap frozen and TFPE specimens, a positive linear relationship was observed between the amount of RNA template and cDNA product. For FFPE specimens, the slope associated with RNA template and cDNA product was greatly reduced, as 1 ug of RNA template from FFPE specimens was necessary to generate the same amount of cDNA as 0.125 ug of RNA template from snap frozen or TFPE specimens. The authors note that while snap frozen and TFPE specimens had similar reverse transcription efficiencies, qRT-PCR analysis of TFPE specimens generated raw Ct values that were 4 cycles higher than that for case-matched snap frozen specimens.
Biospecimens
Preservative Types
- Formalin
- Frozen
- Other Preservative
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Benign
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)
Snap frozen
Xpress Molecular Fixative
Biospecimen Acquisition Biospecimen location Thyroid gland
Kidney
Duodenum
Liver
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
Real-time qRT-PCR Specific Template/input amount 0 ug
0.125 ug
0.25 ug
0.50 ug
1 ug
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Study Purpose
The purpose of this study was to determine if RNA extraction and purification method influenced RNA quality and amplification success in liver adenoma FFPE and TFPE specimens compared to controls snap frozen in isopentane pre-cooled with liquid nitrogen. Differentially preserved specimens were extracted using each method, with the exception of snap frozen specimens and the FFPE extraction kit. Targeted transcripts included GAPDH, beta-2-microglobulin (B2M), and beta actin (ActB).
Summary of Findings:
When RNA was extracted with the Trizol method, distinct ribosomal RNA peaks were observable for snap-frozen but not FFPE or TFPE specimens. In the same samples, 260/280 OD ratios were similar (1.62-1.78) but RINs were much lower in RNA from FFPE (2.1) and TFPE (2.4) specimens compared to those from snap frozen specimens (7.1). Extraction with Trizol followed by purification with the RNeasy kit increased 260/280 OD ratios for all specimens, while extraction and purification with either a miRNA or FFPE kit enriched for small fragments (<200 bp). Importantly, extraction and purification method did not significantly alter RIN or raw Ct values of B2M, beta-actin, or GAPDH, although Ct values were generally higher and more variable for RNA from FFPE (Ct range 32-37) specimens in comparison to snap frozen specimens (Ct range 22-27). In both FFPE and TFPE specimens the ratio of B2M to GAPDH mRNA was altered compared to snap frozen specimens.
Biospecimens
Preservative Types
- Formalin
- Frozen
- Other Preservative
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform 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)
Snap frozen
Xpress Molecular Fixative
Analyte Extraction and Purification Analyte purification None
RNeasy Mini Kit (Qiagen)
RNeasy miRNA Mini Kit (Qiagen)
Analyte Extraction and Purification Analyte isolation method miRNeasy kit (Qiagen)
RNeasy FFPE kit (Qiagen)
Trizol
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
B2M
ActB
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Study Purpose
The purpose of this study was to assess the degree of concordance between differentially preserved liver adenoma specimens, and expression assays. FFPE, TFPE, and control specimens snap frozen in isopentane pre-cooled with liquid nitrogen were analyzed with a Applied Biosytems Whole Genome v 2.0 microarray and by qRT-PCR for GAPDH, B2M, alpha fetoprotein (AFP), interleukin-7 (IL7), and tumor necrosis factor receptor superfamily member 19 (TNFRSF19).
Summary of Findings:
The strength of whole genome microarray correlations were very strong between all technical replicates (0.93-0.97) as well as between RNA from FFPE and TFPE (0.91); however, correlations between RNA from snap-frozen and FFPE (0.47) or TFPE (0.64) were modest. Based upon microarray results, six genes that displayed large differences in expression between fixation methods were also examined by qRT-PCR. When specific transcript levels were quantified in the same sample by qRT-PCR results were similar to those found by microarray for snap frozen specimens but were markedly different for FFPE and TFPE specimens. In fact, expression patterns were generally conserved among snap frozen, FFPE, and TFPE specimens when qRT-PCR results were compared, while expression trends for FFPE and TFPE specimens opposed those for snap frozen specimens for microarray results.
Biospecimens
Preservative Types
- Formalin
- Frozen
- Other Preservative
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform RNA DNA microarray RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Xpress Molecular Fixative
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
B2M
AFP
IL7
TNFRSF19
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Study Purpose
The purpose of the study was to assess the potential impact of time in formalin on qRT-PCR success and efficiency. Specimens were collected from three patients diagnosed with liver adenoma, a patient with colon cancer, and a patient with leiomyosarcoma. Specimens were case-matched and snap frozen in isopentane pre-cooled with liquid nitrogen, or fixed in formalin for different durations prior to paraffin embedding and analysis using the TaqMan Human Molecular Mechanisms of Cancer Array by Applied Biosystems, which contained 92 genes associated with a cancer pathway and 4 control genes.
Summary of Findings:
Amplification of all GAPDH fragment sizes was successful using RNA from snap frozen specimens, while success was dependent upon both time in fixative and amplicon size in case-matched FFPE specimens. Amplification of the longest GAPDH fragment (530 bp) was unsuccessful with RNA from FFPE specimens, and amplification of the 323 bp fragment of GAPDH was only successful in RNA isolated from FFPE specimens fixed for 24 h or less. Raw GAPDH Ct values increased with both amplicon length and time in formalin. A difference of 2-6 Ct was present between the shortest and longest fixation timepoints (4-120 h) for a given amplicon. Specifically, raw Ct values for the 277 bp fragment were 5 cycles lower when specimens were fixed for 4 h compared to 96 h. In general, the magnitude of effect for time in formalin increased with amplicon length. RIN (2.1-2.7) and 260/280 OD ratio (1.92-2.01) were not influenced by time in formalin.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Carcinoma
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer RNA Low density array Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Biospecimen Preservation Time in fixative 4 h
8 h
12 h
24 h
48 h
72 h
96 h
120 h
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
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Study Purpose
The purpose of this study was to determine if paraffin block storage can adversely affect molecular analysis. Case-matched liver adenoma FFPE and control specimens snap frozen in isopentane pre-cooled with liquid nitrogen collected from two patients were analyzed. FFPE blocks were stored for 6 and 12 months prior to analysis using the TaqMan Human Molecular Mechanisms of Cancer Array by Applied Biosystems, which contained 92 genes associated with a cancer pathway and 4 control genes.
Summary of Findings:
RNA from snap frozen controls consistently produced the lowest Ct values for the 92 cancer-related genes targeted by the assay. Compared to RNA from snap frozen controls, Ct values increased for each of the 92 genes by approximately 4 Ct value in RNA from FFPE specimens that were stored for 6 months prior to analysis, and by approximately 8 Ct values when FFPE specimens were stored for 1 year. While the magnitude of effect was gene-specific, variability increased with storage duration.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer RNA Low density array Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Storage Storage duration 6 months
1 year
Real-time qRT-PCR Specific Type of array Human Molecular Mechanisms of Cancer Array
-
Study Purpose
The purpose of this study was to determine if ribosomal RNA (rRNA) is selectively degraded or cross-linked in FFPE and TFPE specimens. FFPE, TFPE, and controls snap frozen in isopentane pre-cooled with liquid nitrogen were analyzed in two liver adenoma cases by amplification of 5 s, 18 s, and 28 s rRNA species by qRT-PCR.
Summary of Findings:
Raw Ct values of short amplicons (50-70 bp) did not differ between RNA isolated from snap frozen, FFPE, and TFPE case-matched specimens for 5 s rRNA, but Ct values for larger rRNA species (18 s and 28 s) did increase in RNA isolated from FFPE and TFPE specimens by 3 and 1.5 Ct values, respectively. The authors conclude that rRNA is fragmented, but present and amplifiable, in FFPE and TFPE specimens.
Biospecimens
Preservative Types
- Formalin
- Other Preservative
- Frozen
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Xpress Molecular Fixative
Real-time qRT-PCR Specific Targeted nucleic acid 5 s rRNA
18 s rRNA
28 s rRNA