Semi-automated purification of total RNA from FFPE tissue was performed using the Maxwell® RSC RNA FFPE Kit (Promega, ref. AS1440) following the manufacturer’s instructions. In brief, FFPE tissue sections were deparaffinized with mineral oil (2 min, 80 °C) and then transferred to 250 µL lysis buffer, centrifuged for phase separation and incubated for 15 min at 56 °C, followed by 1 h at 80 °C. After DNase I treatment (15 min, RT), the aqueous phase was loaded to a Maxwell® RSC Instrument and RNA was semiautomatically isolated by paramagnetic particles. Next, 10 µL MS2 phage control included in the MassARRAY® SARS-CoV-2 Panel (Material and Methods Section 2.3 ) was added to the lysis buffer to monitor RNA extraction.
Maxwell rsc rna ffpe kit
Manufactured by Promega
Sourced in United States, Germany
The Maxwell RSC RNA FFPE Kit is a laboratory equipment product designed to extract and purify RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. It utilizes Promega's Maxwell RSC instrument to automate the RNA extraction process, providing a streamlined and efficient method for obtaining high-quality RNA from challenging FFPE samples.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (21 mentions)
Maxwell rsc instrument, by Promega (13 mentions)
Maxwell rsc dna ffpe kit, by Promega (9 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (9 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Ncounter digital analyzer, by NanoString (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Quantus fluorometer, by Promega (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Taqman 2019 ncov control kit v1, by Thermo Fisher Scientific (2 mentions)
Maxwell rsc simplyrna cells kit, by Promega (2 mentions)
Rna broad range assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorimetric assay, by Thermo Fisher Scientific (2 mentions)
Quantseq 3 mrna seq library prep kit fwd for, by Illumina (2 mentions)
Maxwell rsc simplyrna cells, by Promega (2 mentions)
Ion 550 chip kit, by Thermo Fisher Scientific (2 mentions)
Microm hm 340e, by Thermo Fisher Scientific (2 mentions)
79 protocols using maxwell rsc rna ffpe kit
1
FFPE RNA Extraction using Maxwell RSC
2
FFPE DNA and RNA Extraction and Quantification
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DNA and RNA were extracted from FFPE tissue in accordance to the manufacturer’s protocol (Maxwell® RSC DNA FFPE Kit, AS1450, Maxwell® RSC RNA FFPE Kit, AS1440, Promega, Southampton, UK). The concentrations of DNA and RNA were measured using the Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). All samples were diluted to a final concentration of 10 ng/µl (DNA), 20 ng/µl (RNA). 2 µl RNA was used for reverse transcription (20 µl, Superscript III first-strand synthesis system, Thermo Scientific, Waltham, MA, USA).
3
RNA Isolation from FFPE Tissues
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RNA was isolated from fixed and paraffin-embedded mouse liver samples and from routine clinical tissues. With regards of clinical tissues, two Bouin’s fixed and paraffin-embedded high-grade ovarian cancer samples and ten cancer FFPE samples of different origin were used. Those specimens were collected at the National Cancer Institute of Aviano and at the University Hospital of Trieste. Informed consent was obtained from participants included in the study and ethical approval was obtained by the Institutional Review Board of CRO-Aviano (protocol number 1213, 24 January 2017) and by the ethical committee of the University of Trieste (report n. 17, 4 August 2008).
RNA was isolated from four 10 µm-thick sections of fixed and paraffin-embedded specimens using the Maxwell® RSC instrument (Promega; Madison, WI, USA) following the instruction of the Maxwell® RSC RNA FFPE kit (Cat. No. AS1440). The protocol of isolation includes a digestion step at 56 °C and a de-modification step of 1 h at 80 °C. After RNA isolation, samples were split into aliquots and stored at −80 °C. RNA quantification and purity detection were assessed by NanoDropTM ND-1000 spectrophotometer (Thermo Fischer Scientific; Waltham, MA, USA).
RNA was isolated from four 10 µm-thick sections of fixed and paraffin-embedded specimens using the Maxwell® RSC instrument (Promega; Madison, WI, USA) following the instruction of the Maxwell® RSC RNA FFPE kit (Cat. No. AS1440). The protocol of isolation includes a digestion step at 56 °C and a de-modification step of 1 h at 80 °C. After RNA isolation, samples were split into aliquots and stored at −80 °C. RNA quantification and purity detection were assessed by NanoDropTM ND-1000 spectrophotometer (Thermo Fischer Scientific; Waltham, MA, USA).
4
SARS-CoV-2 Detection from FFPE Tissues
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To detect SARS-CoV-2, RNA was isolated from the embedded tissues using the Maxwell RSC RNA FFPE Kit (Promega, Madison, WI, USA). A TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed by using the TaqMan 2019-nCoV Control Kit v1 (ThermoFisher Scientific, Catalog Number A47533) to target three different viral genomic regions (ORF1AB, S and N genes) and the human RPPH1 gene (RNAse-P). According to the manufacturer’s protocol, a Cт value below 37 in at least two out of three viral genomic regions was considered positive. A case was considered negative if Cт values were above 40. Values between 37 and 40 were considered undetermined and the assay was repeated. Based on the Cт values and quantitation of RPPH1 transcripts, the viral load was calculated as virus genome copy numbers/106RPPH1 transcripts. Samples were always run in duplicates.
5
Detecting Gene Fusions in Tumor RNA
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For RNA sequencing, the tumor RNA was extracted from the paraffin block (tumor fraction: > 90%) with Maxwell® RSC RNA FFPE Kit (Promega, USA). The library was generated with SureSelectXT RNA Direct Kit (Agilent, Santa Clara, USA), and sequenced on an Illumina NovaSeq 6000 at Macrogen (Seoul, Republic of Korea). Raw sequencing reads were analyzed with three kinds of algorithms, namely: DIFFUSE, Fusion catcher, and Arriba (https://github.com/suhrig/arriba/ ), to detect gene fusions. The results were then compared.
Fastq files were briefly aligned by the STAR aligner on the hg19 reference genome for Arriba analysis. The chimeric alignments file and the read-through alignments file were produced, and fusion candidates were generated with a set of filters that detect artifacts based on various characteristic features.
Fastq files were briefly aligned by the STAR aligner on the hg19 reference genome for Arriba analysis. The chimeric alignments file and the read-through alignments file were produced, and fusion candidates were generated with a set of filters that detect artifacts based on various characteristic features.
6
RNA Isolation and Quantification
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RNA-isolation of 1 × 106 cells per sample was performed by using the Maxwell purification platform with appertaining reagents (Maxwell RSC simplyRNA Cells Kit, Promega).
RNA-purification of FFPE specimens was performed by using the Maxwell RSC RNA FFPE Kit (Promega).
The concentration of RNA was determined via fluorometric quantification (Qubit, Thermo Scientific) using the RNA Broad range assay kit according to the manufacturer’s instructions. 1 μl of each isolated RNA sample was applied for measurement.
RNA-purification of FFPE specimens was performed by using the Maxwell RSC RNA FFPE Kit (Promega).
The concentration of RNA was determined via fluorometric quantification (Qubit, Thermo Scientific) using the RNA Broad range assay kit according to the manufacturer’s instructions. 1 μl of each isolated RNA sample was applied for measurement.
7
Detection of DNAJB1-PRKACA Fusion in FFPE Samples
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RNA was extracted from macrodissected 5 µm paraffin sections using the Maxwell® RSC RNA FFPE Kit and the Maxwell® RSC Instrument (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Reverse transcription of RNA and polymerase chain reaction (PCR) of the DNAJB1-PRKACA breakpoint region (forward primer 5′-GTTCAAGGAGATCGCTGAGG-3′, reverse primer 5′- TTCCCGGTCTCCTTGTGTTT-3′) was performed using the QIAGEN OneStep RT-PCR Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). To visualize the detection of the DNAJB1-PRKACA fusion, the PCR product was run on an agarose gel. For sequencing, the PCR product was purified (AMPure, Beckman Coulter, Brea, CA, USA) and aliquots were used for the sequencing reaction with 1 μM of the forward or reverse primer and 2 μl of GenomeLab DTCS-Quick Start Master Mix (Beckman Coulter, Brea, CA, USA) in a final volume of 10 μl according to the manufacturer’s protocol. Sequencing reactions were purified (CleanSEQ, Beckman Coulter, Brea, CA, USA) and analyzed in a GenomeLab GeXP Genetic Analysis System, and evaluated by the GenomeLab GeXP software (Beckman Coulter, Brea, CA, USA).
8
FFPE RNA Extraction and Quality Assessment
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RNA was extracted from 7-μm-thick, mesodissected FFPE tissue sections using the Maxwell® RSC RNA FFPE Kit (Promega) and quantified by Nanodrop 1000 and Qubit (Thermo Fisher Scientific, Waltham, MA, USA) assays. Quality was assessed with the Archer Pre-Seq RNA quality control (QC) qPCR Assay (ArcherDX, Boulder, CO, USA), with a threshold Cq < 31.
9
RNA Extraction from FFPE Tissue
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After routine processing of the tumor specimen, total RNA was isolated from tissue sections with the Maxwell® RSC RNA FFPE Kit (Promega®, Madison, WI, USA) according to the manufacturer’s instructions. RNA was eluted in water and stored at −80 °C until further use. RNA was quantified with the QuantiFluor RNA System on Quantus® Fluorimeter (Promega®) following the “High Standard Calibration” protocol.
10
FFPE RNA Seq Protocol for Illumina NovaSeq
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Total RNA from FFPE from patient's tissues was purified using Maxwell® RSC RNA FFPE Kit (Promega). RNA was quantified using the Qubit 2.0 fluorimetric Assay (Thermo Fisher Scientific). Libraries were prepared from 100 ng of total RNA using the QuantSeq 3′ mRNA‐Seq Library Prep Kit FWD for Illumina (Lexogen GmbH) and sequenced on a 15 NovaSeq 6000 sequencing system using an S1, 100 cycles flow cell (Illumina Inc.). Fastq files were generated using bcl2fastq (version v2.20.0.422, Illumina Inc.), and trimming was performed with bbduk software (bbmap suite 20 37.31, Joint Genome Institute) and alignment on a human genome reference assembly (hg38) with STAR 2.6.0a (Dobin et al, 2013 (link)). Expression levels were determined with htseq‐count 0.9.1 using cellRanger prebuild genes annotations (Single Cell Gene Expression, 10x Genomics; Ensembl Assembly 93). Data normalization was performed using edgeR (Anders et al, 2015 (link)).
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