Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues

Doleshal, Martina; Magotra, Amber A.; Choudhury, Bhavna; Cannon, Brian D.; Labourier, Emmanuel; Szafranska, Anna E.

doi:10.2353/jmoldx.2008.070153

Cited by 208 publications

(167 citation statements)

References 29 publications

(20 reference statements)

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“…Also, it previously was shown that both the RNeasy and RecoverAll kits allow extraction of total RNA of similar integrity and suitable for microarray hybridization. 9,39 Similar findings in both of our cohorts additionally suggest that extraction protocols did not have much influence on the RNA used for library preparation. From this perspective our analysis provided additional independent validation.…”

Section: Discussionsupporting

confidence: 69%

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues

Tyekucheva

Martin

Stack

et al. 2015

The Journal of Molecular Diagnostics

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Archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a readily available but largely untapped resource for gene expression profilingebased biomarker discovery. Several technologies have been proposed to cope with the bias from RNA cross-linking and degradation associated with archival specimens to generate data comparable with RNA from fresh-frozen materials. Direct comparison studies of these RNA expression platforms remain rare. We compared two commercially available platforms for RNA expression profiling of archival FFPE specimens from clinical studies of prostate and ovarian cancer: the Affymetrix Human Gene 1.0ST Array following whole-transcriptome amplification using the NuGen WT-Ovation FFPE System V2, and the NanoString nCounter without amplification. For each assay, we profiled 7 prostate and 11 ovarian cancer specimens, with a block age of 4 to 21 years. Both platforms produced gene expression profiles with high sensitivity and reproducibility through technical repeats from FFPE materials. Sensitivity and reproducibility remained high across block age within each cohort. A strong concordance was shown for the transcript expression values for genes detected by both platforms. We showed the biological validity of specific gene signatures generated by both platforms for both cohorts. Our study supports the feasibility of gene expression profiling and large-scale signature validation on archival prostate and ovarian tumor specimens using commercial platforms. These approaches have the potential to aid precision medicine with biomarker discovery and validation. (J Mol Diagn 2015, 17: 374e381; http://dx

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Section: Discussionsupporting

confidence: 69%

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues

Tyekucheva

Martin

Stack

et al. 2015

The Journal of Molecular Diagnostics

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“…fragmentation, and formation of cross-links between RNA and proteins. Owing to their small size, miRNAs are less prone to degradation, 15,23 therefore, formalin-fixed paraffin-embedded samples are more suitable for miRNA than for mRNA expression analyses. Storage up to 7 years did not cause significant deterioration of miRNA quality, but older samples may exhibit loss of small RNA quality due to oxidation and fixation in nonbuffered formalin.…”

Section: Discussionmentioning

confidence: 99%

MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas

et al. 2010

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MicroRNAs are involved in the pathogenesis of several tumors, however, there have been no data on microRNA expression in pheochromocytomas to date. The objective of our study was to perform microRNA expression profiling in sporadic and hereditary benign, and recurring adrenomedullary tumors. Furthermore, the applicability of formalin-fixed paraffin-embedded tissue samples for the analysis of microRNA expression in pheochromocytomas was examined. MicroRNA expression data of three matched frozen and formalin-fixed paraffin-embedded samples were correlated. A total of 21 formalin-fixed paraffin-embedded samples (sporadic benign, multiple endocrine neoplasia 2, von Hippel-Lindau disease, sporadic recurring) were subjected to microRNA expression profiling using microarrays. MicroRNAs with significant differences in expression were validated and sample sizes were extended including tumors from neurofibromatosis type 1 patients by real-time quantitative reverse-transcription PCR (n ¼ 33). MicroRNA target prediction was carried out by TargetScan and MicroCosm Targets. Pathway analysis of targets was performed by Ingenuity Pathway Analysis and DIANA mirPath. Furthermore, microRNA expression profiles of a malignant pheochromocytoma and a pair of primary and recurrent tumors were studied by TaqMan Human MicroRNA Cards. MicroRNA expression correlated well between frozen and formalin-fixed paraffinembedded samples (70-92%). Microarray analysis revealed 16 significantly differentially expressed microRNAs. Five of these were validated by real-time RT-PCR. miR-139-3p, miR-541 and miR-765 were significantly differentially expressed between sporadic benign and von Hippel-Lindau-related pheochromocytomas. Significantly higher expression of miR-885-5p and miR-1225-3p was found in multiple endocrine neoplasia type 2 and sporadic recurring pheochromocytomas, respectively. Pathway analysis revealed the possible involvement of Notch-and G-proteincoupled receptor signaling in tumor recurrence. MicroRNA expression profiles in the primary recurrent and recurring malignant comparisons have been similar. In conclusion, we have proved that formalin-fixed paraffinembedded samples can be used for the analysis of microRNA expression in pheochromocytomas. MicroRNA expression patterns differ between various sporadic, hereditary and recurring tumors and miR-1225-3p may be useful for identifying recurring pheochromocytomas.

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“…Previously, we identified and validated a superior-performing RNA extraction method allowing isolation of large amounts of quality RNA to perform robust miRNA expression analyses by qRT-PCR in FFPE tissues. 19 Here, we used this method to evaluate miRNA expression profiling in 31 different FFPE tissue blocks up to 11 years old. Using nine different mouse and human tissue types, we demonstrate that the original miRNA composition of frozen tissue is essentially preserved after routine fixation in formalin and long-term storage.…”

Section: Discussionmentioning

confidence: 99%

“…18 The microarray expression levels of selected miRNA species in liver NAT and spleen tissue blocks were confirmed via qRT-PCR using 10 ng of total RNA input and TaqMan microRNA assays (Applied Biosystems, Foster City, CA) as described. 19 …”

Section: Mirna Expression Profilingmentioning

confidence: 99%

Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling

Szafranska

Davison

Shingara

et al. 2008

The Journal of Molecular Diagnostics

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101

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Formalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalinfixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. The same miRNAs were identified as differentially expressed between tissues using both fixed and frozen specimens. While formalin fixation time had only marginal effects on microarray performance, extended storage times for tissue blocks (up to 11 years) resulted in a gradual loss of detection of miRNAs expressed at low levels. Method reproducibility and accuracy were also evaluated in two different tissues stored for different lengths of time. The technical variation between full process replicates, including independent RNA isolation methods, was approximately 5%, and the correlation of expression levels between microarray and real-time quantitative reverse transcriptase polymerase chain reaction was 0.98. Together, these data demonstrate that miRNA expression profiling is an accurate and robust method for the molecular analysis of archived clinical specimens, potentially extending the use of miRNAs as new diagnostic, prognostic, and treatment response biomarkers. (J Mol Diagn

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Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues

Cited by 208 publications

References 29 publications

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues

MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas

Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling

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