Fresh tumor tissue, was collected at intraoperative pathology evaluation, diced into pieces of 1–2mm diameter, stirred, and randomly assigned to: 1) RNAlater solution later stored at −80°C freezer (FF); or 2) 10% neutral buffered formalin and paraffin-embedded as a FFPE tissue block [10 (link)]. Phenotypically, the nine breast cancers were defined by pathologic status of hormone receptors (HR) and HER2 receptor as: HR+/HER2− in five, HR+/HER2+ in one, and triple receptor-negative (TN) in three. RNA was purified from FF samples using the RNeasy Mini Kit (Qiagen, Valencia, CA), and FFPE samples from 10µm sections using High Pure FFPE RNA Isolation Kit (Roche, Indianapolis, IN). A DNase-I treatment step was included in both.
High pure ffpe rna isolation kit
Manufactured by Roche
Sourced in Germany, United States, Switzerland
The High Pure FFPE RNA Isolation Kit is a laboratory equipment designed to extract and purify RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit utilizes a specialized, optimized protocol to facilitate the recovery of high-quality RNA from challenging FFPE samples, making it suitable for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop one, by Thermo Fisher Scientific (4 mentions)
Trusight rna fusion panel, by Illumina (3 mentions)
Miniseq sequencer, by Illumina (3 mentions)
Qubit rna xr assay kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Agilent bioanalyzer, by Agilent Technologies (2 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nsolver 4.0 analysis software, by NanoString (2 mentions)
Ncounter prep station, by NanoString (2 mentions)
Masterpure complete dna and rna purification kit, by Illumina (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nsolver analysis software, by NanoString (1 mentions)
Ncounter analysis system, by NanoString (1 mentions)
Ncounter pancancer pathways panel, by NanoString (1 mentions)
Truseq rna access kit, by Illumina (1 mentions)
Tetro cdna synthesis kit, by Meridian Bioscience (1 mentions)
Allprep dna rna ffpe isolation kit, by Qiagen (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
24 protocols using high pure ffpe rna isolation kit
1
Breast Cancer Tissue Preservation and RNA Extraction
2
Evaluation of BRAF and RAS Mutations in Thyroid Neoplasms
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Archival thyroid neoplasm that had been surgically removed between 2010 and 2014 at Konkuk University Medical Center were blindly re-evaluated according to the 2004 World Health Organization classification of thyroid neoplasm by the two pathologists (Tae Sook Hwang, who is an endocrine pathologist, and Young Sin Ko). In case of a disagreement and to reach a consensus, another endocrine pathologist (Chan-Kwon Jung) independently reviewed the cases. Of the 600 classical PTC cases selected, 518 had BRAF mutation and 6 had RAS mutation. Finally, 76 BRAF and RAS wild-type classical PTC cases were selected. Tumor portion on the hematoxylin and eosin-stained FFPE tissue slides was marked by the pathologist, and total RNA was isolated from two-to-three FFPE tissue sections (10 μm thick) using High Pure FFPE RNA isolation kit (Roche Diagnostics). RT-PCR was performed, and Ct values of the RET/PTC1 rearrangement and GAPDH expression were evaluated. The Ct value defining the analysis as positive is greater than 40 cycles.
3
Gene Expression Profiling of Soft Tissue Tumors
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The level of gene expression was measured using the NanoString nCounter analysis system (NanoString Technologies, Seattle, WA, USA), as described previously [20 (link)]. Seven cases of icSFT/HPC and six cases of exSFT/HPC were subjected to the gene expression assay. For better RNA quality, the most recently acquired cases were selected from each group based on the test time point. Total RNA was extracted from 10-μm-thick FFPE sections using the High Pure FFPE RNA Isolation Kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer’s instructions. Total RNA was hybridized with preset probes and evaluated using the NanoString nCounter digital analyzer (PhileKorea Technologies, Seoul, Korea). The nCounter pan-cancer pathways panel included 770 genes from 13 canonical pathways (NanoString Technologies). Data analysis was performed using nSolver analysis software (NanoString Technologies) and the mRNA profiling data were normalized using housekeeping genes. Normalized data were log2-transformed for further analysis.
4
Prostate Cancer RNA Profiling Protocol
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The H&E slides from all samples were reviewed by two pathologists to mark the areas with the collected sections of 63 prostatectomy specimens composed of 10 PCAs with low Gleason score (≤7, Low GS), 10 PCAs with high Gleason score (>7, High GS), five benign prostate tissues as a control for fold change, and thyroidal follicular carcinoma showing PPAR-γ nuclear staining confirmed by immunohistochemistry as positive control. Subsequently, 10 µm thick sections cut from the formalin-fixed, paraffin-embedded blocks for dissecting the target area from the slides, then, transferred into microcentrifuge tubes. Before RNA isolations, deparaffinization was performed with 1 mL xylene with 10 min incubation time, and 1 mL absolute ethanol for 10 min. Total RNA was extracted by using High Pure FFPE RNA Isolation Kit (Roche Applied Science, Penzberg, Germany), according to the manufacturers' instructions. RNA yield and quality was assessed by measuring the ratio of spectrophotometric absorbance (260 nm/230 nm and 260 nm/280 nm) using NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The extracted RNA samples were considered to be acceptable only with absorbance ratio between 1.0 and 2.0 at 260 nm/230 nm, and between 1.8 and 2.0 at 260 nm/280 nm.
5
Extracting RNA from FFPE Menisci
6
Profiling FFPE Tumor RNA with NanoString
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The RNA of 22 tumors was obtained from FFPE tissue. Five 5-µm thick sections were cut from the FFPE blocks and, by following the manufacturer’s instructions, total RNA was obtained with the High Pure FFPE RNA Isolation Kit (Roche, Basel, Switzerland). RNA concentrations were measured using the Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples with adequate concentrations were hybridized to the nCounter® PanCancer IO 360TM Gene Expression Panel (NanoString, Seattle, WA, USA), containing 770 genes, for 16 h using a thermocycler. The samples were transferred to the nCounter Prep Station (NanoString, Seattle, WA, USA) for further processing. The gene expression profiles of the samples were digitalized with the nCounter Digital Analyzer. The results were quantified using nSolver 4.0 Analysis Software (NanoString, Seattle, WA, USA).
7
Breast Cancer Sample Preparation
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Fresh tumor tissue was collected at intraoperative pathology evaluation, diced into pieces of 1- to 2-mm diameter, stirred, and randomly assigned to RNAlater solution later stored in a −80°C freezer (FF) or 10% neutral buffered formalin and paraffin-embedded as an FFPE tissue block.10 (link) Phenotypically, the nine breast cancers were defined by the pathologic status of hormone receptors (HRs) and human epidermal growth factor receptor 2 (HER2) as HR-positive/HER2-negative in five, HR-positive/HER2-positive in one, and triple receptor-negative in three. RNA was purified from FF samples using the RNeasy Mini Kit (Qiagen, Valencia, CA) and FFPE samples from 10-μm sections using High Pure FFPE RNA Isolation Kit (Roche, Indianapolis, IN). A DNase-I treatment step was included in both.
8
Targeted RNA-seq of Cancer Fusions
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RNA was isolated from FFPE samples using five 10 μm thick tissue sections. A High Pure FFPE RNA Isolation Kit (Roche, Indianapolis, IN) was used according to the manufacturer’s protocol. RNA was quantified using a Nanodrop apparatus and evaluated with an Agilent 2100 bioanalyzer.
Next-generation sequencing-based targeted RNA-sequencing analysis was performed using the Illumina TruSight RNA Fusion Panel and a MiniSeq sequencer according to the manufacturer’ s recommendation (Illumina, San Diego, CA). This targeted RNA fusion panel consists of 507 of the most well-known cancer-related fusion genes (including TFE3), and the gene list is available atwww.illumina.com . The TruSight RNA Fusion Panel covers 7690 exonic regions that are targeted with a total of 21,283 probes.
Next-generation sequencing-based targeted RNA-sequencing analysis was performed using the Illumina TruSight RNA Fusion Panel and a MiniSeq sequencer according to the manufacturer’ s recommendation (Illumina, San Diego, CA). This targeted RNA fusion panel consists of 507 of the most well-known cancer-related fusion genes (including TFE3), and the gene list is available at
9
Detecting RET/PTC1 Rearrangement in PTC
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PTC cells from the archival FNA slides from the Department of Pathology, Konkuk University Medical Center were used. The slides that were selected were from samples aspirated from ten thyroid nodules with histopathological diagnosis of classical-type PTC. Study approval was obtained from the Institutional Review Board (KUH1210043). After the coverslips were removed from the slides, approximately 100 atypical follicular cells were dissected with a 26-gauge needle under the light microscope, and total RNA was extracted using MasterPure Complete DNA and RNA Purification Kit (Epicentre). RT-PCR was performed, and Ct values of the RET/PTC1 rearrangement and GAPDH expression were evaluated. Tumor portion on the hematoxylin and eosin-stained FFPE tissue slides was marked by the pathologist, and total RNA was isolated from two-to-three FFPE tissue sections (10 μm thick) using High Pure FFPE RNA isolation kit (Roche Diagnostics). RT-PCR was performed and Ct values of the RET/PTC1 rearrangement and GAPDH expression were evaluated. The Ct values defining the analysis as positive is greater than 40 cycles.
10
Glomerular Transcriptome Profiling from FFPE Kidney Biopsies
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Formalin-fixed and paraffin-embedded (FFPE) sections of stored kidney biopsy tissue were microdissected, as previously reported [16 (link)], to obtain glomerular cross-sections. RNA was extracted and purified from enzymatically digested specimens using the High Pure FFPE RNA Isolation Kit® (Roche Molecular Systems, Inc., Pleasanton, CA, USA) according to previously described techniques [20 (link)]. RNA libraries were generated using the TruSeq RNA Access kit (Illumina Inc.,San Diego, CA, USA) and sequenced (75 base-pair paired-end RNAseq) according to protocols previously utilized by our group [21 (link),22 (link)]. Gene expression data are available in the GEO repository through accession number GSE216841.
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