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

2008 ◽  
Vol 10 (3) ◽  
pp. 203-211 ◽  
Author(s):  
Martina Doleshal ◽  
Amber A. Magotra ◽  
Bhavna Choudhury ◽  
Brian D. Cannon ◽  
Emmanuel Labourier ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Extraction Methods ◽  
Microrna Expression ◽  
Total Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Rna Extraction Methods ◽  
Formalin Fixed ◽  
Total Rna Extraction

scholarly journals Impact of RNA Extraction and Target Capture Methods on RNA Sequencing Using Formalin-Fixed, Paraffin Embedded Tissues

2019 ◽  
Author(s):  
Christopher A. Hilker ◽  
Aditya V. Bhagwate ◽  
Jin Sung Jang ◽  
Jeffrey G Meyer ◽  
Asha A. Nair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Seq ◽  
Gene Detection ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

AbstractFormalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

Quantitative determination of predictive cancer biomarkers in formalin-fixed, paraffin-embedded tissue using a new, fully automated method for RNA isolation

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 11032-11032
Author(s):  
B. M. Müller ◽  
R. Kronenwett ◽  
G. Hennig ◽  
K. Weber ◽  
K. Bohmann ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Cancer Biomarkers ◽  
Ffpe Tissue ◽  
Rt Pcr ◽  
Total Rna ◽  
Tissue Sections ◽  
Automated Method ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

11032 Background: Personalized cancer therapy depends on the evaluation of tissue-based biomarkers in routine tumor samples. As many new biomarkers are measured on the mRNA level, standardized procedures for mRNA analysis in formalin-fixed, paraffin-embedded (FFPE) tissue are needed. In this study, we present a novel, standardized, and fully automated method for fast isolation of total RNA from FFPE tissue sections. Methods: Tissue samples (n = 501) from 167 breast carcinomas which had been stored between 2 months and 21 years were investigated. Total RNA was extracted from tissue sections using a new method based on silica-coated iron oxide beads in combination with a specific liquid-handling robot. Novel and unique features of the method are an automated extraction-integrated deparaffinization step and a bead-based negative selection step to eliminate any undigested tissue. Yield of RNA was assessed using the Ribogreen assay. mRNA fragment lengths were estimated by reverse transcription PCR (RT-PCR) for G6PDH. Expression of the breast cancer biomarkers ESR1, PGR and HER2 was measured by kinetic RT-PCR (kPCR) and compared with immunohistochemistry (IHC). Results: RNA was successfully isolated from all samples, with a mean yield of 1.4 μg/sample (range: 0.1–7.8 μg) and fragment lengths of at least 150 bp in 99% of samples. PCR analyses could be performed in all samples. Investigating three sections of each tumor, we observed a low section-to-section variability of kPCR results (root of mean squared errors of relative ESR1, PGR or HER2 expression in three sections: 0.2–0.5 Ct values). Comparing kPCR results with IHC, we detected a good concordance between both methods, with agreements of 98.4% (standard error [SE]: 2.7%) for ESR1, 84.4% (SE: 5.3%) for PGR and 89.9% (SE: 8.9%) for HER2. Conclusions: This novel RNA extraction method is a major technical improvement for implementation of reproducible, high-throughput and cost-efficient testing of cancer biomarkers in the clinical routine and in gene-expression research studies using archived FFPE material in molecular labs. [Table: see text]

scholarly journals Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yulia Newton ◽  
Andrew J. Sedgewick ◽  
Luis Cisneros ◽  
Justin Golovato ◽  
Mark Johnson ◽  
...  
Keyword(s):  
Large Scale ◽  
Clinical Decision Making ◽  
Rna Extraction ◽  
Transcriptome Profiling ◽  
Clinical Decision ◽  
Extraction Methods ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

Abstract Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

2011 ◽  
Vol 77 (18) ◽  
pp. 6476-6485 ◽  
Author(s):  
Zhanbei Liang ◽  
Ann Keeley
Keyword(s):  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Direct Detection ◽  
Rna Extraction ◽  
Milk Powder ◽  
Extraction Methods ◽  
Content Type ◽  
Total Rna ◽  
Rna Extraction Methods

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

scholarly journals Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

2005 ◽  
Vol 17 (6) ◽  
pp. 574-578 ◽  
Author(s):  
Ming Y. Deng ◽  
He Wang ◽  
Gordon B. Ward ◽  
Tammy R. Beckham ◽  
Thomas S. McKenna
Keyword(s):  
Real Time ◽  
Classical Swine Fever Virus ◽  
Rna Extraction ◽  
Classical Swine Fever ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcription Pcr ◽  
Rna Extraction Methods ◽  
Positive Results

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

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