Quantstudio 3d instrument

Manufactured by Thermo Fisher Scientific

Sourced in United States

The QuantStudio™ 3D Instrument is a digital PCR system designed for high-precision, quantitative analysis of nucleic acid samples. The instrument utilizes a microfluidic chip to partition samples into thousands of individual reaction chambers, enabling precise measurement of target DNA or RNA molecules.

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QuantStudio 3D Digital PCR Instrument

6 protocols using quantstudio 3d instrument

Real-Time and Digital PCR for Gene Expression

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Extraction of RNA was performed from VECs and VICs using the Total RNA Purification Plus Kit (Norgen Biotek Corp.). RNA was quantified using Nanodrop and used for two steps PCR amplification with TaqMan Reverse Transcription Reagent kit (Life Technologies). Total RNA (1 μg) was converted into cDNA. Real Time PCR (qPCR) was performed on ABI Prism 7900 HT (Applied Biosystems), according to the manufacturer’s instructions and analysis were performed using software SDS2.4 (Life Technologies). Digital PCR (dPCR) was performed on a QuantStudio™ 3D Digital PCR System platform composed by the QuantStudio™ 3D Instrument, the Dual Flat Block GeneAmp® PCR System 9700 and the QuantStudio™ 3D Digital PCR Chip Loader (Life Technologies). dPCR was performed according to the manufactures’s instructions and analysis were executed with QuantStudio® 3D AnalysisSuite™ (Life Technologies). Primers labelled with FAM® dyes were used to evaluate the expression of target genes, while for housekeeping genes primers labelled with VIC® dye were implemented. For normalization we used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ribosomal protein large P0 (RPLP0) gene expression levels and the primers used are listed in Supplemental Table 3

Songia P., Branchetti E., Parolari A., Myasoedova V., Ferrari G., Alamanni F., Tremoli E, & Poggio P. (2016). Mitral Valve Endothelial Cells Secrete Osteoprotegerin During Endothelial Mesenchymal Transition. Journal of molecular and cellular cardiology, 98, 48-57.

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Digital PCR Analysis of POLR1D and ERBB2

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SCNAs of POLR1D and ERBB2 were analyzed using digital PCR (dPCR) and performed on the QuantStudio 3D platform (Life Technologies, Carlsbad, CA, USA). Pre-designed TaqMan assays specific for the detection of the copy number of POLR1D (Hs02926936_cn), ERBB2 (Hs00450668_cn), and a reference assay (TERT; 4403315) were purchased from Thermo Fisher. For dPCR, a total amount of 3–5 ng plasma DNA was used as input and samples were run in duplicate using the QuantStudio™ 3D Digital PCR 20 K Chip Kit v2 and a QuantStudio 3D instrument (Life Technologies, Carlsbad, CA, USA). Raw data were analyzed using the relative quantification module of the QuantStudio 3D Analysis Suite Software, including a Poisson correction. The confidence level was set to 95%, and the desired precision value was 10%.

Zhou Q., Perakis S.O., Ulz P., Mohan S., Riedl J.M., Talakic E., Lax S., Tötsch M., Hoefler G., Bauernhofer T., Pichler M., Gerger A., Geigl J.B., Heitzer E, & Speicher M.R. (2020). Cell-free DNA analysis reveals POLR1D-mediated resistance to bevacizumab in colorectal cancer. Genome Medicine, 12, 20.

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Quantifying GPCR Expression via Digital PCR

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To measure GPCR expression, HEKn were grown to 50% confluence and collected for RNA extraction. Digital PCR was performed on a QuantStudio 3D Digital PCR System platform consisting of a ProFlex PCR machine (including a chip adapter kit), an automatic chip loader, and a QuantStudio 3D Instrument (Life Technologies, Carlsbad, CA, USA). Specific TaqMan probes for digital PCR (Supplementary Table 1; see Supplementary Information) were selected from the TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA, USA).

Sayama K., Yuki K., Sugata K., Fukagawa S., Yamamoto T., Ikeda S, & Murase T. (2021). Carbon dioxide inhibits UVB-induced inflammatory response by activating the proton-sensing receptor, GPR65, in human keratinocytes. Scientific Reports, 11, 379.

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Quantitative detection of gene mutations

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dPCR was performed on a QuantStudio™ 3D Digital PCR System platform consisting of a Gene Amp 9700 PCR machine (including a chip adapter kit), an automatic chip loader, and the QuantStudio™ 3D Instrument (Thermo Fisher Scientific). Consequently, the collected data were analyzed with QuantStudio 3D AnalysisSuite Cloud Software (Thermo Fisher Scientific). Mutation analysis of cfDNA by dPCR was based on a 5′‐exonuclease assay using TaqMan®‐MGB probes targeting KRAS G12V, G12D, G12R, and GNAS R201C (Thermo Fisher Scientific, Catalog number: A44177), which were chosen according to the genetic profiles observed in tissue samples.

Takano S., Fukasawa M., Shindo H., Takahashi E., Fukasawa Y., Kawakami S., Hayakawa H., Kuratomi N., Kadokura M., Maekawa S, & Enomoto N. (2021). Digital next‐generation sequencing of cell‐free DNA for pancreatic cancer. JGH Open: An Open Access Journal of Gastroenterology and Hepatology, 5(4), 508-516.

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Chip-based Digital PCR Analysis of miRNAs

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Chip-based dPCR was performed on a QuantStudio 3D Digital PCR System platform composed of the QuantStudio 3D Instrument (Thermo Fisher Scientific; 4489084), the Dual Flat Block GeneAmp PCR System 9700 (Thermo Fisher Scientific; 4484078), and the QuantStudio 3D Digital PCR Chip Loader (Thermo Fisher Scientific; 4482592). dPCR was performed according to the manufacturer’s instructions. In particular, enzyme activation was performed at 96 °C for 10 min, denaturation at 98 °C for 30 s, followed by annealing/extension at 56 °C for 2 min (40 cycles), and then final extension at 60 °C for 2 min. Analysis was executed with the online version of the QuantStudio 3D AnalysisSuite (Thermo Fisher Cloud, Waltham, MA, USA). The evaluated copy numbers for each miRNA are expressed as the mean of Log10 of the copy number/µL within a 95% confidence interval (CI). Upper and lower limits of the 95% CI are indicated within square brackets.
Primers and probes purchased from Thermo Fisher Scientific were labelled with FAM dyes and used to evaluate the expression of candidate miRNAs (Supplementary Table S1).

D’Alessandra Y., Valerio V., Moschetta D., Massaiu I., Bozzi M., Conte M., Parisi V., Ciccarelli M., Leosco D., Myasoedova V.A, & Poggio P. (2022). Extraction-Free Absolute Quantification of Circulating miRNAs by Chip-Based Digital PCR. Biomedicines, 10(6), 1354.

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Digital PCR Analysis of Tumor Mutations

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Digital polymerase chain reaction (PCR) was performed on a QuantStudio™ 3D Digital PCR System platform composed of a Gene Amp 9700 PCR machine (including a chip adapter kit), an automatic chip loader, and the QuantStudio™ 3D Instrument (Thermo Fisher Scientific). Consequently, the collected data were analyzed with QuantStudio 3D AnalysisSuite Cloud Software (Thermo Fisher Scientific). Mutation analysis in dPCR was based on a 5′‐exonuclease assay using TaqMan®‐MGB probes targeting KRAS G12V, G12D, G12A, G12S, G12C, G13D, Q61R, TP53 R248W, Y126*, Y107*, R158H, V272M, R175H, G244D, G245D, BRAF V600E, and CDKN2A H66R (Thermo Fisher Scientific, Catalog number: A44177). These targets were selected based on the mutations detected in tissues by NGS as indicated below, and one of the tissue‐derived mutations was selected for dPCR analysis of cfDNA.

Ishida Y., Takano S., Maekawa S., Yamaguchi T., Yoshida T., Kobayashi S., Iwamoto F., Kuno T., Hayakawa H., Matsuda S., Fukasawa M., Shindo H., Inoue T., Nakayama Y., Ichikawa D., Sato T, & Enomoto N. (2020). Fractionated small cell‐free DNA increases possibility to detect cancer‐related gene mutations in advanced colorectal cancer. JGH Open: An Open Access Journal of Gastroenterology and Hepatology, 4(5), 978-986.

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