Absolutely rna ffpe kit

Manufactured by Agilent Technologies

Sourced in United States

The Absolutely RNA FFPE Kit is a laboratory equipment product designed for the extraction and purification of total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit provides the necessary reagents and protocols to efficiently isolate high-quality RNA from FFPE samples for downstream applications.

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Lab products found in correlation

High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Sureselect human all exon v4 utrs kit, by Agilent Technologies (1 mentions) Quant it picogreen dsdna reagent and kit, by Thermo Fisher Scientific (1 mentions) Hiseq 2000, by Illumina (1 mentions) Rneasy ffpe kit, by Agilent Technologies (1 mentions) Qpcr human reference total rna, by Agilent Technologies (1 mentions) Taqman probe based gene expression assay, by Thermo Fisher Scientific (1 mentions) Rotor gene 6000, by Qiagen (1 mentions) 7500 sequence detector, by Thermo Fisher Scientific (1 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions) Tripure isolation reagent, by Roche (1 mentions) Victorxtm4 microplate reader, by PerkinElmer (1 mentions) High capacity rna to dna kit, by Thermo Fisher Scientific (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions)

8 protocols using absolutely rna ffpe kit

Exome Sequencing and Genotyping for NSCLC

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The Absolutely RNA FFPE kit (modified protocol for DNA extraction, Agilent Technologies, Santa Clara, CA) was used to prepare the DNA. Using 1 μg of dsDNA, quantified by Quant-iT PicoGreen dsDNA Reagent and Kits (Life Technologies, Carlsbad, CA), the exome-sequencing libraries were prepared. All exomes were captured using the SureSelect Human All Exon V4+UTRs Kit (Agilent Technologies) (71 mb). The exome capture libraries were sequenced by HiSeq 2000 (Illumina, San Diego, CA) to generate 100-bp paired-end data.
The Illumina HumanOmniExpress-FFPE BeadChip assay was used to analyze the genotype, DNA copy number, and loss of heterozygosity (LOH) in 48 primary-normal paired samples. All samples, except for 1 (n = 47), passed our quality control metrics for sample identity and data quality. A subset of 693,000 high-quality single nucleotide polymorphisms was selected for all analyses (Supplemental Figure 1, Supplemental Digital Content 2, http://links.lww.com/JTO/A626). A gene was considered copy number amplified if the calculated copy number in a sample was more than or equal to 4, and a gene was considered copy loss if the copy number in a sample was 0. Recurrent genomic regions with DNA copy gain and loss were identified using GISTIC, version 2.0.^{14 (link),15 (link)}

Umemura S., Mimaki S., Makinoshima H., Tada S., Ishii G., Ohmatsu H., Niho S., Yoh K., Matsumoto S., Takahashi A., Morise M., Nakamura Y., Ochiai A., Nagai K., Iwakawa R., Kohno T., Yokota J., Ohe Y., Esumi H., Tsuchihara K, & Goto K. (2014). Therapeutic Priority of the PI3K/AKT/mTOR Pathway in Small Cell Lung Cancers as Revealed by a Comprehensive Genomic Analysis. Journal of Thoracic Oncology, 9(9), 1324-1331.

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Complementary DNA Synthesis from RNA

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Approximately one hundred samples of “normal”-appearing mucosa and one hundred samples of tumoral mucosa were included in the study. Complementary DNA (cDNA) was synthesized from up to 2 µg of RNA using the High Capacity cDNA Reverse Transcription (RT) kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Briefly, in a 20 µL reaction mix, 2.0 µL of 10X RT Buffer, 0.8 µL of 25X dNTP Mix (100 mM), 2.0 µL of 10X RT Random Primers, 1.0 µL of MultiScribe Reverse Transcriptase, 4.2 µL of nuclease-free water, and 10.0 µL of RNA were used. The RT conditions were as follows: Annealing at 25 °C for 10 min, DNA polymerization at 37 °C for 120 min, and enzyme deactivation at 85 °C for 5 min.
All RT reactions included one no-template negative control. Moreover, 1 µL containing 1 µg of the QPCR Human Reference Total RNA, part of the Absolutely RNA FFPE Kit (Agilent, Santa Clara, CA, USA), was used as a positive control to monitor the quality of the RT.

Lopes C., Pereira C., Farinha M., Medeiros R, & Dinis-Ribeiro M. (2020). Prostaglandin E2 Pathway Is Dysregulated in Gastric Adenocarcinoma in a Caucasian Population. International Journal of Molecular Sciences, 21(20), 7680.

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Tumor Cell RNA Extraction from FFPE

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An H&E stain from each specimen was obtained and reviewed by a pathologist to accurately select blocks with a high percentage of tumor cells and normal tissue. Only tumor samples containing more than 60% of tumor tissue using the H&E-stained section were analyzed. Three to five adjacent unstained slides of 10 μm were obtained from the corresponding block for the extraction of total RNA. Total RNA was extracted from FFPE tissue sections with a Qiagen RNeasy FFPE Kit and from OCT tissues sections with an Agilent Absolutely RNA FFPE Kit, following the manufacturers’ protocols. The concentration of RNA was assessed.

Karim N.A., Ullah A., Pathrose P., Fathallah H., Perry A., Morris J.C., Wang J, & Starnes S.L. (2022). Does Molecular Profiling of KRAS-Mutant Non-Squamous Non-Small Cell Lung Cancer (NSCLC) Help in Treatment Strategy Planning?. Current Oncology, 29(7), 4779-4790.

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GC Mucosa Transcriptome Analysis

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Approximately one hundred samples of “normal”-appearing mucosa and tumoral mucosa of GC patients were randomly selected.
Complementary DNA (cDNA) was synthesized from up to 2 µg of RNA using the High-Capacity cDNA Reverse Transcription (RT) kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.
All RT reactions included one no-template negative control. Moreover, 1 µL containing 1 µg of the QPCR Human Reference Total RNA, part of the Absolutely RNA FFPE Kit (Agilent, Santa Clara, CA, USA), was used as a positive control to monitor the quality of the RT.

Lopes C., Pereira C., Farinha M., Medeiros R, & Dinis-Ribeiro M. (2021). Genetic Variations in Prostaglandin E2 Pathway Identified as Susceptibility Biomarkers for Gastric Cancer in an Intermediate Risk European Country. International Journal of Molecular Sciences, 22(2), 648.

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RNA Isolation and qPCR for FPN1 Expression

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Isolation of total RNA was performed with the Absolutely RNA FFPE kit (Agilent Technologies, California, USA), according to the manufacturers’ protocol. Briefly, sections from each archival sample were deparaffinized and incubated overnight with a lysis buffer containing proteinase K and submitted to a series of washes-on-column until elution. Immediately after, 50 ng of RNA were reversed transcribed with Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) in a total volume of 20 μL, according to the manufacturer’s protocol. Evaluation of FPN1 mRNA levels (Hs00221783_m1) was performed in a Rotor-Gene 6000 instrument (Qiagen, CA, USA) with a TaqMan® Probe-based gene expression assay (Applied Biosystems, CA, USA). Reactions were carried out in triplicate and gene expression levels calculated relative to GUSB mRNA levels (Hs99999908_m1). Mean relative expression was calculated based on the formula ΔCt = Ct target gene—Ct endogenous control gene and fold change on 2^{(ΔCt breast tumor samples – ΔCt normal breast samples)}.

Marques O., Porto G., Rêma A., Faria F., Cruz Paula A., Gomez-Lazaro M., Silva P., Martins da Silva B, & Lopes C. (2016). Local iron homeostasis in the breast ductal carcinoma microenvironment. BMC Cancer, 16, 187.

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CD44 Isoform Expression Analysis

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TriPure isolation reagent (Roche Diagnostics GmbH) was used to extract total RNA from BC cells, while RNA from tissues was extracted using the Absolutely RNA FFPE Kit (Agilent), according to the manufacturer's instructions. Complementary DNA (cDNA) reverse transcription and mRNA expression were performed as previously described 30 (link). The relative expression of total CD44 and its isoforms was determined by RT-PCR analysis using TaqMan Gene Expression Assays (total CD44: Hs01075864_m1; CD44e5-v2: Hs01075866_m1; CD44e5-v3: Hs01081480_m1; CD44e5-v8: Hs01081475_m1, CD44e5-e15: Hs01081473_m1; Applied Biosystems) in a 7500 Sequence Detector (Applied Biosystems). β-2-microglobulin (B2M) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT) were used for normalization, also as previously described. Details on TaqMan Gene Expression Assays can be found in Table S1. All samples were run in duplicate and relative mRNA gene expression was calculated with the 2^-ΔCt formula.

Gaiteiro C., Soares J., Relvas-Santos M., Peixoto A., Ferreira D., Paulo P., Brandão A., Fernandes E., Azevedo R., Palmeira C., Freitas R., Miranda A., Osório H., Prieto J., Lima L., Silva A.M., Santos L.L, & Ferreira J.A. (2022). Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion. Theranostics, 12(7), 3150-3177.

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Intracellular ROS Detection via H2DCF-DA

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Detection of intracellular reactive oxygen species (ROS) were reached using H2DCF-DA [2,7dichlorodihydro uorescein diacetate (10mM)] as probe. Brie y, cells were treated for 48h and after that, the incubation medium was replaced by Balanced Salt Solution (BSS -NaCl 124 mM, KCl 5.8 mM, dextrose 10 mM, Hepes 20 mM, CaCl2(H2O)2 0.3 mM) and cells were incubated for 30 minutes at 37°C.
At the end of the incubation time H2DCF-DA 20 μM were added and cells were incubated for 45 minutes at 37°C.The uorescence signal was quanti ed using a microplate uorescence reader (VictorXTM4 microplate reader, PerkinElmer Inc, Waltham, Ma, USA), using 485 nm as excitation and 535 nm as emission wavelength.
mRNA analysis in real time PCR Total cytoplasmic RNA from cell lines was extracted using the TRIzol reagent protocol and RNA from FFPE tissues was extracted using the Absolutely RNA FFPE Kit (Agilent, CA, USA) using the manufacturer's instructions. RNA concentration was measured using the Qubit™ RNA HS Assay Kit (Thermo Fisher Scienti c). Aliquots (1 µg) of RNA were converted to cDNA using random hexamer primers. Quantitative changes in mRNA levels were estimated by real time PCR using the following cycling conditions: 35

Chiavari M., Ciotti G.M.P., Canonico F., Altieri F., Navarra P., & Lisi L. (2020). PDIA3 Reduce Glioma-associated Macrophage/microglia Pro-tumor Activation trough IL-6-STAT3-PDIA3 Pathway.

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Verification of Novel Fusion Transcripts

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Novel fusions not previously reported in the literature were verified by reverse transcription (RT)-PCR and Sanger sequencing. RNA was extracted from formalin-fixed, paraffinembedded (FFPE) blocks using the Absolutely RNA FFPE kit (Agilent, Santa Clara, CA), and converted to cDNA using the High Capacity RNA-to-DNA Kit (Thermo Fisher Scientific, Waltham, MA). Custom primers were designed for each fusion transcript (see Supplemental Table S1 for primer sequences) (Integrated DNA Technologies, Coralville, IA). Nested primers were designed in some instances where initial PCR was unsuccessful. PCR was performed in 20-mL reactions using 20 ng of cDNA, 10 mL of KAPA Hi-Fi Hotstart Polymerase ReadyMix (Roche, Basel, Switzerland), and 0.5 mL each forward and reverse primer. The PCR program was as follows: 95 C for 3 minutes, then 40 cycles of 98 C for 30 seconds, 60 C for 30 seconds, and 72 C for 30 seconds; followed by 72 C for 5 minutes. Resulting PCR products were gel electrophoresed and visualized to verify correct band size. PCR products were purified using the QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany) and Sanger sequenced (Gen-eWiz, South Plainfield, NJ). Sanger sequencing results were compared with NGS fusion transcripts.

Sun L., McNulty S.N., Evenson M.J., Zhu X., Robinson J.A., Mann P.R., Duncavage E.J, & Pfeifer J.D. (2021). Clinical Implications of a Targeted RNA-Sequencing Panel in the Detection of Gene Fusions in Solid Tumors. The Journal of molecular diagnostics : JMD, 23(12).

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