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Expressart ffpe clear rna ready kit

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The ExpressArt FFPE Clear RNA Ready kit is a laboratory product designed to extract high-quality RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit provides a streamlined process for RNA extraction, purification, and preparation for downstream applications such as gene expression analysis.

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Lab products found in correlation

Trusight rna fusion panel, by Illumina (32 mentions) Agilent bioanalyzer, by Agilent Technologies (11 mentions) Miseq platform, by Illumina (7 mentions) Qubit rna hs assay kit, by Thermo Fisher Scientific (6 mentions) Amsbio s, by AMS Biotechnology (3 mentions) Rna seq libraries, by Illumina (2 mentions) Bioanalyzer rna 6000 nano kit, by Agilent Technologies (2 mentions) Trusight rna pan cancer panel, by Illumina (1 mentions) Nextseq 500 system, by Illumina (1 mentions) Miseq v3, by Illumina (1 mentions) Fusionplex custom solid panel, by Illumina (1 mentions) Rna 6000 chip, by Agilent Technologies (1 mentions)

33 protocols using expressart ffpe clear rna ready kit

1

RNA Extraction and Library Preparation for RNA-seq

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Scrolls obtained from formalin-fixed paraffin-embedded tissue blocks were cut (4 at 10 microns) into Eppendorf tubes. RNA was extracted using ExpressArt FFPE Clear RNA Ready kits (Amsbio, Cambridge, MA). Cases 1–2, and 4: RNA-seq libraries were prepared with the TruSight RNA Fusion Panel (Illumina, San Diego, CA), as previously described.11 (link) Cases 3 and 5: libraries were prepared using the Archer FusionPlex™ assay (Enzymatics Inc, Beverly, MA), as previously described.12 (link)
Han R., Dermawan J.K., Demicco E.G., Ferguson P.C., Griffin A.M., Swanson D., Antonescu C.R, & Dickson B.C. (2022). ZFP64::NCOA3 GENE FUSION DEFINES A NOVEL SUBSET OF SPINDLE CELL RHABDOMYOSARCOMA. Genes, chromosomes & cancer, 61(11), 645-652.
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2

RNA-seq Analysis of Formalin-Fixed Tissue

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Scrolls (3–4 at 10 microns) of formalin-fixed paraffin-embedded tissue blocks were cut into Eppendorf tubes. Cases 1–2: RNA was extracted using ExpressArt FFPE Clear RNA Ready kits (Amsbio, Cambridge, MA), and RNA-seq libraries prepared using 20–100 ng RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA). Cases were sequenced with 76 base-pair paired-end reads on an Illumina MiSeq with 8 samples per flow cell (~3 million reads per sample). Analysis was performed using both the STAR aligner and Manta fusion caller, and the JAFFA fusion caller utilizing BOWTIE2 aligner;9 (link),10 (link) Case 3: RNA-seq libraries were prepared with the TruSight RNA Pan-Cancer Panel (Illumina, San Diego, CA). Sequencing with 76 base-pair paired-end reads on an Illumina NextSeq 500 System (Illumina, San Diego, CA). Analysis was performed using the STAR aligner and STAR fusion caller11 (link) Case 4 was confirmed by Caris Life Sciences, (Irving, TX).
Lacambra M.D., Antonescu C.R., Chit C., Chiu W.K., Demicco E.G., Ferguson P.C., Swanson D., To K.F., Zhang L, & Dickson B.C. (2022). EXPANDING THE SPECTRUM OF MESENCHYMAL NEOPLASMS WITH NR1D1-REARRANGEMENT. Genes, chromosomes & cancer, 61(7), 420-426.
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3

RNA-Seq of FFPE Tissue for Fusion Detection

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Formalin-fixed paraffin-embedded tissue blocks were cut into scrolls (3-4 at 10 microns) and placed in Eppendorf tubes. ExpressArt FFPE Clear RNA Ready kits (Amsbio, Cambridge, MA) were used to extract RNA. The RNA-seq libraries were prepared using 20-100 ng RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA). Each sample was sequenced with 76 base-pair paired-end reads on an Illumina MiSeq, with 8 samples per flow cell (~3 million reads per sample). Cases 1, and 3-4 were analyzed using both the STAR aligner and Manta fusion caller, and the JAFFA fusion caller utilizing BOWTIE2 aligner;11 (link), 12 (link) Case 2 was analyzed using the STAR aligner and STAR fusion caller.13 (link)
Lacambra M.D., Weinreb I., Demicco E.G., Chow C., Sung Y.S., Swanson D., To K.F., Wong K.C., Antonescu C.R, & Dickson B.C. (2019). PRRX-NCOA1/2 REARRANGEMENT CHARACTERIZES A DISTINCTIVE FIBROBLASTIC NEOPLASM. Genes, chromosomes & cancer, 58(10), 705-712.
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4

FFPE RNA Extraction and RNA-seq Analysis

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RNA was extracted from formalin-fixed paraffin-embedded tissue. Tissue sections were either derived from blocks cut into scrolls (3–4 at 10 microns) into Eppendorf tubes, or blocks cut onto glass slides (7–10 at 4 microns) and later scrapped into Eppendorf tubes. RNA was extracted using ExpressArt FFPE Clear RNA Ready kits (Amsbio, Cambridge, MA). RNA-seq libraries were prepared using 20–100 ng RNA and the TruSight RNA Fusion Panel (Illumina, San Diego, CA). Each sample was sequenced with 76 base-pair paired-end reads on an Illumina MiSeq, with 8 samples per flow cell (~3 million reads per sample). Analysis was done using both the STAR aligner and Manta fusion caller, and the JAFFA fusion caller and BOWTIE2 aligner respectively.8 (link),9 (link)
Dickson B.C., T-S Chung C., Hurlbut D.J., Marrano P., Shago M., Sung Y.S., Swanson D., Zhang L, & Antonescu C.R. (2019). Genetic Diversity in Alveolar Soft Part Sarcoma: A Subset Contain Variant Fusion Genes, Highlighting Broader Molecular Kinship with Other MiT Family Tumors. Genes, chromosomes & cancer, 59(1), 23-29.
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5

FFPE RNA Extraction and Fusion Gene Detection

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Briefly, for each case scrolls (3–4 at 10 microns) were cut from formalin-fixed paraffin-embedded tissue into Eppendorf tubes. RNA was extracted using the ExpressArt FFPE Clear RNA Ready kit (Amsbio, Cambridge, MA). RNA-seq libraries were prepared using 20-100 ng total RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA), an enrichment-based assay that targets 507 known fusion-associated genes. Each sample was sequenced with 76 base-pair paired-end reads on an Illumina MiSeq at 8 samples per flow cell (~3 million reads per sample). The results were analyzed using the STAR aligner and Manta fusion caller as well as the JAFFA fusion caller utilizing BOWTIE2 aligner.(15 (link), 16 (link))
The mRNA expression levels of NUTM1, and the respective fusion partners, were evaluated and compared to those of other samples analyzed on the same targeted RNA sequencing platform.
Dickson B.C., Sung Y.S., Rosenblum M.K., Reuter V.E., Harb M., Wunder J.S., Swanson D, & Antonescu C.R. (2018). NUTM1 GENE FUSIONS CHARACTERIZE A SUBSET OF UNDIFFERENTIATED SOFT TISSUE AND VISCERAL TUMORS. The American journal of surgical pathology, 42(5), 636-645.
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6

Targeted RNA Sequencing of FFPE Samples

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Four cases were analyzed by targeted RNA sequencing, using RNA extracted from FFPE tissue with the Amsbio’s ExpressArt FFPE Clear RNA Ready kit (Amsbio LLC, Cambridge, MA). The fragment length was assessed with an RNA 6000 chip on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using 20 to 100 ng total RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA). Targeted RNA sequencing was performed on an Illumina MiSeq platform. Reads were independently aligned with STAR (version 2.3) against the human reference genome (hg19) and analyzed by STAR-Fusion.
Suurmeijer A.J., Dickson B.C., Swanson D., Sung Y.S., Zhang L, & Antonescu C.R. (2020). Variant WWTR1 Gene Fusions in Epithelioid Hemangioendothelioma – A Genetic Subset Associated with Cardiac Involvement. Genes, chromosomes & cancer, 59(7), 389-395.
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7

FFPE RNA Sequencing and Fusion Assay

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Formalin-fixed paraffin-embedded tissue sections (either scrolls [3–4 at 10 microns] or tissue scraped from glass slides [4–5 at 4 microns] were obtained from each case. RNA extraction was performed with the ExpressArt FFPE Clear RNA Ready kit (Amsbio, Cambridge, MA). Libraries were prepared using 20–100 ng total RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA), an enrichment-based assay targeting 507 fusion-associated genes. RNA sequencing (RNA-seq) was performed with 76 base-pair paired-end reads on an Illumina MiSeq at 8 samples per flow cell (~3 million reads per sample). The results were analyzed using both the STAR aligner and Manta fusion caller, and the BOWTIE2 aligner and JAFFA fusion caller (10 (link), 11 (link)).
Archer™ FusionPlex™ technology was used to develop the MSK-Solid Fusion assay, which is a clinical molecular diagnostic essay performed in a CLIA-accredited laboratory utilizing multiplex polymerase chain reaction (PCR) to detect oncogenic fusion transcripts involving 62 genes as described previously (12 (link)).
Dickson B.C., Antonescu C.R., Demicco E.G., Leong I., Anderson N.D., Swanson D., Zhang L., Fletcher C.D, & Hornick J.L. (2021). Hybrid Schwannoma-Perineurioma Frequently Harbors VGLL3 Rearrangement. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 34(6), 1116-1124.
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8

Targeted RNA Sequencing of Fusion Genes

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RNA was extracted from FFPE tissue using Amsbio’s ExpressArt FFPE Clear RNA Ready kit (Amsbio LLC, Cambridge, MA). Fragment length was assessed with an RNA 6000 chip on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using 20–100 ng total RNA with the Trusight RNA Fusion Panel (Illumina, San Diego, CA), which targets a list of N genes of interest. Each sample was subjected to targeted RNA sequencing on an Illumina MiSeq at 8 samples per flow cell (approximately 3 million reads per sample). All reads were independently aligned with STAR (ver 2.3) and BowTie2 against the human reference genome (hg19) for Manta-Fusion and TopHat-Fusion analysis, respectively, for fusion discovery. The mRNA expression levels of certain genes of interest (BCOR, SS18L1, SSX1, etc.) were evaluated and compared to those of other samples analyzed in the same batch of targeted RNA sequencing platform, including 3 fusion-negative URCS, 2 epithelioid hemangioendotheliomas, one SS with SS18-SSX2 fusion, and one inflammatory myofibroblastic tumor.
Kao Y.C., Sung Y.S., Zhang L., Kenan S., Singer S., Tap W.D., Swanson D., Dickson B.C, & Antonescu C.R. (2017). BCOR Upregulation in a Poorly Differentiated Synovial Sarcoma with SS18L1-SSX1 Fusion – A Pathologic and Molecular Pitfall. Genes, chromosomes & cancer, 56(4), 296-302.
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9

RNA-seq of FFPE Sarcoma Samples

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RNA was extracted from formalin-fixed paraffin-embedded tissue cut onto positively charged glass slides (four μm section, 7–10 per case) using the ExpressArt FFPE Clear RNA Ready kit (Amsbio, Cambridge, MA). RNAseq libraries were prepared using 20–100 ng total RNA with the TruSight RNA Fusion Panel (Illumina, San Diego, CA), an enrichment-based assay that targets 507 known fusion-associated genes. The sample was sequenced with 76 base-pair paired-end reads on an Illumina MiSeq. Results were analyzed using the STAR aligner and Manta fusion caller as well as the JAFFA fusion caller utilizing BOWTIE2 aligner (25 (link), 26 (link)). mRNA expression level of NR4A3 was evaluated in the index case and compared to >100 sarcoma types available on the same RNAseq platform. The expression of PGR could not be evaluated since this gene was not represented on the targeted RNAseq gene list.
Chiang S., Samore W., Zhang L., Sung Y.S., Turashvili G., Murali R., Soslow R.A., Hensley M.L., Swanson D., Dickson B.C., Stewart C.J., Oliva E, & Antonescu C.R. (2019). PGR Gene Fusions Identify a Molecular Subset of Uterine Epithelioid Leiomyosarcoma with Rhabdoid Features. The American journal of surgical pathology, 43(6), 810-818.
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10

FFPE RNA Fusion Panel Sequencing

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RNA was extracted from FFPE tissue using Amsbio’s ExpressArt FFPE Clear RNA Ready kit (Amsbio LLC, Cambridge, MA). The case was tested on the TruSight RNA Fusion Panel (Illumina, San Diego, CA), using 100 ng total RNA for RNA-sequencing libraries preparation. Fragment length was assessed with an RNA 6000 chip on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). The sample was subjected to targeted RNA sequencing on an Illumina MiSeq (~3 million reads per sample). All reads were independently aligned with STAR (version 2.3) and BowTie2 against the human reference genome (hg19) for Manta-Fusion and TopHat-Fusion analysis, respectively. The details of this methodology are as previously described4 (link).
Torrence D., Zhang L., Sung Y.S., Dickson B, & Antonescu C.R. (2021). Hyalinizing epithelioid tumors with OGT-FOXO fusions. A case report of a non-acral soft tissue mass harboring a novel FOXO4 gene rearrangement. Genes, chromosomes & cancer, 60(7), 498-503.
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