Qiaamp dna ffpe tissue kit

Manufactured by Qiagen

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The QIAamp DNA FFPE Tissue Kit is a laboratory equipment designed for the purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. It is used to extract high-quality genomic DNA from FFPE samples for downstream applications such as PCR, sequencing, and other molecular biology techniques.

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QIAamp kit (104 citations) QIAamp Tissue Kit (88 citations) QIAamp FFPE Tissue Kit (51 citations) FFPE Tissue Kit (21 citations) FFPE DNA Kit (17 citations) QIAamp DNA FFPE Tissue (13 citations) QIAamp DNA FFPE (11 citations) DNA kits (7 citations) FFPE Kits (2 citations)

1 693 protocols using qiaamp dna ffpe tissue kit

Genomic DNA Extraction from FFPE Tissues

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Biopsy tissue sections were adjusted to room temperature for 20 min, deparaffinised in xylene (20 min), and rehydrated in EtOH (99% EtOH for 20 min, 96% EtOH for 10 min, and 70% EtOH for 3 min). Tissue sections were scraped into 1.5 ml tubes and genomic DNA extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) with the following modifications: Deparaffination was performed as described above and incubation in ATL buffer with proteinase K at 56 °C was extended from 1 to 16 hours. Genomic DNA was extracted from macrodissected fresh-frozen and FFPE prostatectomy specimens using the PUREGENE DNA purification kit (Gentra systems) with proteinase K treatment, and the miRNeasy FFPE kit (Qiagen)/RNeasy plus mini kit (Qiagen), respectively, as described previously21 (link)54 (link). In addition, FFPE prostatectomy specimens from 4 PC patients were used for laser microdissection (LMD). Tissue sections (5 μm) were mounted on polyethylene naphthalate membrane glass, deparaffinised and rehydrated in xylene/EtOH, and stained with a 0.25% w/v solution of cresyl violet (Sigma-Aldrich) in 99.99% EtOH. The Veritas Microdissection Instrument (Arcturus) was used for LMD of individual DAN/PAN/CAN areas from 10–20 serial sections and genomic DNA extracted using the Allprep DNA/RNA FFPE Kit (Qiagen) or the QIAamp DNA FFPE Tissue Kit (Qiagen).

Møller M., Strand S.H., Mundbjerg K., Liang G., Gill I., Haldrup C., Borre M., Høyer S., Ørntoft T.F, & Sørensen K.D. (2017). Heterogeneous patterns of DNA methylation-based field effects in histologically normal prostate tissue from cancer patients. Scientific Reports, 7, 40636.

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Genomic Profiling of FFPE Tumors

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Formalin-fixed, paraffin-embedded (FFPE) samples were used to produce 5-mm-thick slides. The pathologist evaluated the hematoxylin and eosin (HE) slides of each specimen and ensured that the tumor content was more than 20%. DNA from FFPE tumor tissues and matched blood was obtained by using QIAamp DNA FFPE Tissue Kit and QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany), respectively. Agarose gel electrophoresis was used to examine the quality and integrity of the DNA.
Genomic DNA was prepared for paired-end library construction using a QIAamp DNA FFPE Tissue Kit and QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany). The libraries were captured by using the NGS-based WES of OrigiMed (Shanghai, China). Targeted genomic regions were sequenced with an Illumina NovaSeq 6000.

Xie L., Cai Z., Lu H., Meng F., Zhang X., Luo K., Su X., Lei Y., Xu J., Lou J., Wang H., Du Z., Wang Y., Li Y., Ren T., Xu J., Sun X., Tang X, & Guo W. (2023). Distinct genomic features between osteosarcomas firstly metastasing to bone and to lung. Heliyon, 9(5), e15527.

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Simultaneous DNA and RNA Extraction

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Cells were pelleted by centrifugation, and DNA and RNA were extracted using a protocol adapted from QIAGEN AllPrep DNA/RNA FFPE Kit (Cat# 80234), QIAamp DNA FFPE Tissue Kit (Cat# 56404) and miRNeasy FFPE kit (Cat# 217504). Briefly, cell pellets were resuspended in 240 μL Buffer PKD and 16 μL proteinase K (QIAGEN # 80234), lysed by vortexing, and centrifuged 20 min (15-30 min) at > 20,000 x g (room temperature). The supernatant was transferred to a new 1.5 mL centrifuge tube for RNA extraction, and the pellet was reserved for DNA extraction. The supernatant was incubated at 80°C for 15 min (on a thermal mixer at 300 rpm), and then centrifuged at 14,000 rpm for 2 min. The supernatant was transferred to a new 2.0 mL centrifuge tube and RNA was extracted using the miRNeasy FFPE kit (QIAGEN cat# 217504) according to the manufacturer’s instructions. The pellet containing the DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN cat# 56404) according to the manufacturer’s instructions. Both RNA and DNA kits utilize spin columns to wash and then elute nucleic acids.

Huang D., Chowdhury S., Wang H., Savage S.R., Ivey R.G., Kennedy J.J., Whiteaker J.R., Lin C., Hou X., Oberg A.L., Larson M.C., Eskandari N., Delisi D.A., Gentile S., Huntoon C.J., Voytovich U.J., Shire Z.J., Yu Q., Gygi S.P., Hoofnagle A.N., Herbert Z.T., Lorentzen T.D., Calinawan A., Karnitz L.M., Weroha S.J., Kaufmann S.H., Zhang B., Wang P., Birrer M.J, & Paulovich A.G. (2021). Multiomic analysis identifies CPT1A as a potential therapeutic target in platinum-refractory, high-grade serous ovarian cancer. Cell Reports Medicine, 2(12), 100471.

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BRAF Mutation Analysis of CD1a+ Histiocytes

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Tumor DNA was extracted from formalin‐fixed, paraffin‐embedded tissues from our pathological sample bank. Following HE and immunohistochemical staining, the samples with the highest CD1a‐positive histiocyte density were selected for further BRAF mutation analysis.
DNA was extracted from the collected tissue samples using the QIAGEN QIAamp DNA FFPE Tissue Kit (154051332, QIAGEN China (Shanghai) Co. Ltd., Shanghai, China), following the manufacturer's protocol.

Huang H., Lu T., Sun Y., Li S., Li J., Xu K., Feng R.E, & Xu Z.J. (2019). Association between clinicopathologic characteristics and BRAFV600E expression in Chinese patients with Langerhans cell histiocytosis. Thoracic Cancer, 10(10), 1984-1992.

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Microdissection and DNA Extraction from FFPE Tissue

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Tumour cell rich regions, identified by examination of the HE-stained sections and matching the regions on the 10 μm consecutive tissue sections, were micro-dissected from three slides, and same-specimen samples were pooled, and DNA extracted using Qiagen DNA deparaffinisation solution, followed by processing using a Qiagen QIAamp DNA FFPE Tissue Kit (Qiagen, Germantown, MD, USA). The sample yield, purity and quality of the eluted DNA were assessed using a Qubit dsDNA BR Assay Kit and Qubit Fluorometer (ThermoFisher, Waltham, MA, USA) and the Agilent QC-Plex multiplex PCR assay (Agilent, Santa Clara, CA, USA), respectively. For samples to be sequenced, DNA yield was at least 40ng per sample and DNA Quality Coefficient (DQC), as determined by Agilent Qcplex, was at least 0.34. Sample DCQ ranged between 0.34 and 1.88.

Cain S.A., Pope B., Mangiola S., Mantamadiotis T, & Drummond K.J. (2023). Somatic mutation landscape in a cohort of meningiomas that have undergone grade progression. BMC Cancer, 23, 216.

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DNA Extraction from Tissue Samples

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DNA was extracted from tissue samples using the QIAGEN QIAamp DNA FFPE Tissue Kit (56404, QIAGEN) following the manufacturer’s protocol and in 50 μl of buffer ATE (included in the kit). The absorbance of the extracted DNA was measured using a Merinton SMA4000spectrophotometer (Merinton Inc., Beijing, China), and the DNA was diluted with distilled water to a final concentration of approximately 2–3 ng/μl.

Sun J., Zhang J., Lu J., Gao J., Ren X., Teng L., Duan H., Lin Y., Li X., Zhang B, & Liang Z. (2016). BRAF V600E and TERT Promoter Mutations in Papillary Thyroid Carcinoma in Chinese Patients. PLoS ONE, 11(4), e0153319.

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Tumor DNA Isolation from FFPE Samples

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Tumor DNA was isolated by first having two pathologists independently evaluate H&E-stained 5 μm tumor sections in order to confirm tumor histopathology. Tumor-rich paraffin-embedded sections (>70% tumor tissue) were then selected, and stromal sections were trimmed away using H&E-stained sections for guidance. The remaining tumor tissue was then transferred into a lysis buffer and DNA was isolated based on directions provided with the QIAGEN QIAamp DNA FFPE Tissue Kit (Cat no. 56404, Qiagen, Shanghai, China). A 50 μL volume of ATE buffer was employed for DNA sample elution.

Liu Z., Gu Y., Yu F., Zhou L., Cheng X., Jiang H., Huang Y., Zhang Y., Xu T., Qian W, & Li X. (2022). The Number of Intraoperative Intestinal Venous Circulating Tumor Cells Is a Prognostic Factor for Colorectal Cancer Patients. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 4162354.

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BRAF and NRAS Mutation Analysis in Primary Melanoma

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Each FFPE block with confirmed diagnosis of primary invasive melanoma was recut for four 10-µm sections for DNA extraction. These sections were deparaffinized using xylene and purified with ethanol (100%). Qiagen QIAamp DNA FFPE Tissue Kit (Qiagen, Strasse, Germany) was used for DNA extraction and quantified using NanoDrop Spectrophotometer (ThermoScientific, Wilmington, DE). PCR was performed using Promega GoTaq Flexi DNA Polymerase (Promega, Madison, WI) and 10x Buffer A from Fisher Scientific Taq DNA Polymerase (Fisher Scientific, Fair Lawn, NJ). Amplified PCR products were checked for quality with 1% agarose gels (Fisher Scientific, Fair Lawn, NJ). Pyrosequencing of the BRAF and NRAS mutations was performed using the Qiagen PyroMark Q24 platform (Qiagen, Strauss, Germany). Based on the pyrosequencing results, each of the 127 tumors was assigned one of the following genotype: BRAF^V600E mutation, 31 patients (24.4%); NRAS^Q61R mutation, 31 patients (24.4%); and wild-type at both loci, 72 patients (56.7%). Among tumors with mutations, 7 tumors (5.5%) had mutations at both loci. For analytic purposes, the 7 patients with both mutations were examined separately, and therefore the final analysis included 120 subjects.

Wu S., Kuo H., Li W.Q., Canales A.L., Han J, & Qureshi A.A. (2014). Association between BRAFV600E and NRASQ61R Mutations and Clinicopathologic Characteristics, Risk Factors and Clinical Outcome of Primary Invasive Cutaneous Melanoma. Cancer causes & control : CCC, 25(10), 1379-1386.

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Tumor DNA Extraction from FFPE Tissue

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Sections (5 μm thick) were cut from paraffin-embedded tumor tissue blocks and stained with H&E for histopathological examination. Each sample was evaluated by two experienced pathologists. To obtain maximal tumor DNA, we chose tumor-rich paraffin block specimens whose tumor components were greater than 70% and the amount of stroma was less than 30%. For DNA isolation, 5-μm-thick sections were used for each case. The H&E section was used as a reference, and tumor-rich regions of the sections were trimmed off from the slides based on their respective H&E staining patterns and transferred to lysate buffer (included in the DNA purification kit). DNA in the collected tissue samples was extracted using the QIAGEN QIAamp DNA FFPE Tissue Kit (Cat No. 56404, Qiagen, Shanghai, China) following the manufacturer’s protocol. DNA from each sample was eluted in 50 μl of ATE buffer (included in the kit).

Zhang J., Zheng J., Yang Y., Lu J., Gao J., Lu T., Sun J., Jiang H., Zhu Y., Zheng Y., Liang Z, & Liu T. (2015). Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases. Scientific Reports, 5, 18678.

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DNA Methylation Analysis by Pyrosequencing

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Specific polymerase chain reaction (PCR) primers were designed to target the screened methylation sites, and the genomic DNA of tumor FFPE samples was extracted using the QIAGEN Qiaamp DNA FFPE Tissue Kit (QIAGEN, 56404). The DNA was then methylated using the QiagenEpitect Bisulfite Kit (Qiagen, 59104). The methylation site primers were designed using PyroMark Assay Design 2.0. The DNA was amplified using PCR and then detected via pyrosequencing.

Wang X., Jia J., Gu X., Zhao W.W., Chen C., Wu W., Wang J, & Xu M. (2021). Screening of Breast Cancer Methylation Biomarkers Based on the TCGA Database. International Journal of General Medicine, 14, 9833-9839.

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