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Hs dsdna kit qbit

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The HS dsDNA kit Qbit is a fluorescence-based quantification solution for measuring the concentration of double-stranded DNA (dsDNA) in a sample. It provides a sensitive and accurate method for quantifying low amounts of dsDNA.

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Lab products found in correlation

Qiaamp dna micro kit, by Qiagen (3 mentions) Qiaamp dna ffpe tissue kit, by Qiagen (2 mentions) Smart seq2 protocol, by Illumina (2 mentions) Hiseqv4, by Illumina (2 mentions) Quantseq 3 mrna seq kit, by Lexogen (2 mentions) Sense mrna seq library kit, by Lexogen (2 mentions) Rnaeasy mini kit, by Qiagen (2 mentions)

6 protocols using hs dsdna kit qbit

1

FFPE DNA Extraction and Sequencing

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The DNA was extracted from FFPE tissue using the QIAamp DNA FFPE Tissue Kit (QIAGEN). The extracted DNA was quantified by fluorometry (HS dsDNA Kit Qbit, Thermo Fisher Scientific). A detailed description of the methods used for sequencing and copy number analysis can be found in Supplemental Methods. Individual sequence information was deposited in the National Center for Biotechnology Information’s database of Genotypes and Phenotypes, study phs002437.v1.p1: https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs002437.v1.p1
Gutkind J.S., Molinolo A.A., Wu X., Wang Z., Nachmanson D., Harismendy O., Alexandrov L.B., Wuertz B.R., Ondrey F.G., Laronde D., Rock L.D., Rosin M., Coffey C., Butler V.D., Bengtson L., Hsu C.H., Bauman J.E., Hewitt S.M., Cohen E.E., Chow H.H., Lippman S.M, & Szabo E. (2021). Inhibition of mTOR signaling and clinical activity of metformin in oral premalignant lesions. JCI Insight, 6(17), e147096.
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2

FFPE and LCM DNA Extraction

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The DNA was extracted from FFPE tissue using the QIAamp DNA FFPE tissue kit and QIAamp DNA Micro Kit (Qiagen) for the test specimen or LCM specimen, respectively. For the LCM sample, the membrane and adhering tissue were peeled off the caps using a razor blade and the peeled membrane was incubated in proteinase K digestion reaction overnight for 16 h at 56 °C to maximize DNA yield after cell lysis and the elution was done in 20 µL. The extracted DNA was quantified by fluorometry (HS dsDNA kit Qbit—Thermofisher). All samples used in the study are described in Additional file 1: Table S1.
Nachmanson D., Steward J., Yao H., Officer A., Jeong E., O’Keefe T.J., Hasteh F., Jepsen K., Hirst G.L., Esserman L.J., Borowsky A.D, & Harismendy O. (2020). Mutational profiling of micro-dissected pre-malignant lesions from archived specimens. BMC Medical Genomics, 13, 173.
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3

Extracting DNA from Membrane Tissue

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The membrane and adhering tissue were peeled off the caps using a razor blade and the peeled membrane was incubated in proteinase K digestion reaction overnight for 16 h at 56 °C to maximize DNA yield after cell lysis The DNA was extracted using the QIAamp DNA Micro Kit (Qiagen) and the elution was done in 20 µL. The extracted DNA was quantified by fluorometry (HS dsDNA kit Qbit—Thermofisher).
Nachmanson D., Officer A., Mori H., Gordon J., Evans M.F., Steward J., Yao H., O’Keefe T., Hasteh F., Stein G.S., Jepsen K., Weaver D.L., Hirst G.L., Sprague B.L., Esserman L.J., Borowsky A.D., Stein J.L, & Harismendy O. (2022). The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ. NPJ Breast Cancer, 8, 6.
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4

Laser-Capture Microdissection and DNA Extraction

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Matching regions from 6 adjacent sections were collected on capsure Macro Cap (for DNA, N=3 slides) or HS caps (for RNA, N=3 slides), region size, and unambiguous match permitting.
Post-dissection, all caps were covered and stored at -20ºC with desiccant. DNA extraction and QC: The membrane and adhering tissue were peeled off the caps using a razor blade and the peeled membrane was incubated in proteinase K digestion reaction overnight for 16 h at 56°C to maximize DNA yield after cell lysis The DNA was extracted using the QIAamp DNA Micro Kit (Qiagen) and the elution was done in 20 µL. The extracted DNA was quantified by fluorometry (HS dsDNA kit Qbit -Thermofisher).
Nachmanson D., Officer A., Mori H., Gordon J., Evans M., Steward J.S., Yao H., O’Keefe T.J., Hasteh F., Stein J.L., Jepsen K., Weaver D.L., Hirst G.L., Sprague B.L., Esserman L.J., Borowsky A.D., Stein J.L., & Harismendy O. (2021). The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ.
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5

Bulk and Single-Cell RNA-Seq Protocols

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Whole RNA was isolated from cells using RNAeasy Mini Kit (Qiagen). For RNA isolation from bulk tumours, tumours were homogenized in Trizol and RNA was extracted using chloroform, followed by a clean-up step using the RNAeasy Mini Kit. For standard RNA-seq, libraries were generated using the Lexogen sense mRNA seq library kit using 500 ng – 1 μg of input RNA, for Quant-seq libraries were generated using the Lexogen Quant-seq 3’ mRNA seq kit using 500 ng input RNA, both according to manufacturer’s instructions. All libraries were quality controlled on a fragment analyser and quantified using the Qbit HS dsDNA kit (Thermo Scientific). Smart-seq analysis of 100 sorted DCs (DAPI- CD45+ CD11c+ CD103+) or 100 sorted T cells (DAPI- CD45+ CD3e+ CD8a+) was performed in-house using the Smart-seq2 protocol by Illumina59 (link). Library prep was performed according to manufacturer’s instruction using the Nextera kit after direct sorting into RNA lysis buffer. Sequencing was performed in-house on Illumina HiSeqV4 to obtain 50 bp single-end sequencing reads.
Haas L., Elewaut A., Gerard C.L., Umkehrer C., Leiendecker L., Pedersen M., Krecioch I., Hoffmann D., Novatchkova M., Kuttke M., Neumann T., de Silva I.P., Witthock H., Cuendet M.A., Carotta S., Harrington K.J., Zuber J., Scolyer R.A., Long G.V., Wilmott J.S., Michielin O., Vanharanta S., Wiesner T, & Obenauf A.C. (2021). Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumour microenvironment and cross-resistance to immunotherapy in melanoma. Nature cancer, 2(7), 693-708.
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6

Bulk and Single-Cell RNA-Seq Protocols

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Whole RNA was isolated from cells using RNAeasy Mini Kit (Qiagen). For RNA isolation from bulk tumours, tumours were homogenized in Trizol and RNA was extracted using chloroform, followed by a clean-up step using the RNAeasy Mini Kit. For standard RNA-seq, libraries were generated using the Lexogen sense mRNA seq library kit using 500 ng – 1 μg of input RNA, for Quant-seq libraries were generated using the Lexogen Quant-seq 3’ mRNA seq kit using 500 ng input RNA, both according to manufacturer’s instructions. All libraries were quality controlled on a fragment analyser and quantified using the Qbit HS dsDNA kit (Thermo Scientific). Smart-seq analysis of 100 sorted DCs (DAPI- CD45+ CD11c+ CD103+) or 100 sorted T cells (DAPI- CD45+ CD3e+ CD8a+) was performed in-house using the Smart-seq2 protocol by Illumina59 (link). Library prep was performed according to manufacturer’s instruction using the Nextera kit after direct sorting into RNA lysis buffer. Sequencing was performed in-house on Illumina HiSeqV4 to obtain 50 bp single-end sequencing reads.
Haas L., Elewaut A., Gerard C.L., Umkehrer C., Leiendecker L., Pedersen M., Krecioch I., Hoffmann D., Novatchkova M., Kuttke M., Neumann T., de Silva I.P., Witthock H., Cuendet M.A., Carotta S., Harrington K.J., Zuber J., Scolyer R.A., Long G.V., Wilmott J.S., Michielin O., Vanharanta S., Wiesner T, & Obenauf A.C. (2021). Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumour microenvironment and cross-resistance to immunotherapy in melanoma. Nature cancer, 2(7), 693-708.
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