Cell line miRNAs were isolated from cell pellets using the miRNeasy kit (Qiagen) according to the manufacturer's instructions. Experiments were carried out in singleplex using the QuantStudio 3D‐Digital PCR System platform using the QuantStudio 3D‐Digital PCR Master Mix V2 (Thermo Fisher Scientific), according to the manufacturer's instructions. A negative control was included in each analysis. The target gene miR‐29b was analyzed in triplicate for both samples and controls. We prepared 15 μL of reaction mix containing 7.5 μL of 2× QuantStudio 3D Digital PCR Master Mix (Life Technologies), 0.75 μL of 20× TaqMan‐MGB‐FAM‐probe assay, 5.0 μL diluted cDNA (50 ng/μL), and 1.75 μL nuclease‐free water (Qiagen). Loaded chips underwent thermocycling following specific amplification conditions: 96°C for 8 min, 40 cycles at 60°C for 2 min, and 98°C for 30 s, followed by a final extension step at 60°C for 2 min. The expression levels of miR‐29b were determined using a quantification analysis module on Thermo Fisher Cloud (Thermo Fisher Scientific).
Quantstudio 3d digital pcr system platform
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio™ 3D Digital PCR System is a high-throughput digital PCR platform for precise quantification of nucleic acids. It utilizes microfluidic technology to partition samples into thousands of individual reaction wells, enabling absolute quantification of target molecules. The system provides a sensitive and accurate method for detecting and quantifying low-abundance targets, such as rare mutations or copy number variations.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 3d instrument, by Thermo Fisher Scientific (3 mentions)
Quantstudio 3d analysissuite cloud software, by Thermo Fisher Scientific (2 mentions)
Gene amp 9700 pcr machine, by Thermo Fisher Scientific (2 mentions)
Taqman mgb probe, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d digital pcr master mix, by Thermo Fisher Scientific (2 mentions)
Thermo fisher cloud, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr master mix v2, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by Qiagen (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Dual flat block geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr chip loader, by Thermo Fisher Scientific (1 mentions)
Primers and probe, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr 20k chip, by Thermo Fisher Scientific (1 mentions)
6 protocols using quantstudio 3d digital pcr system platform
1
Quantitative Analysis of miR-29b in Cells
2
Quantitative detection of gene mutations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
3
Chip-based Digital PCR Analysis of miRNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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 ).
Primers and probes purchased from Thermo Fisher Scientific were labelled with FAM dyes and used to evaluate the expression of candidate miRNAs (
4
Assessing Tumor Mutation Status from cfDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We determined the specificity and sensitivity of our assay by analysing archival serum and plasma cfDNA from 40 cancer patients at presentation attending the Quiron Dexeus University Hospital (33 NSCLC, 2 CRC and 5 metastatic melanoma) with paired tumour tissue. In addition, we tested archival serum and plasma cfDNAs from 12 responder patients and 11 patients at the time of tumour progression after treatment (18 NSCLC, 2 CRC and 3 metastatic melanoma; Table 1 ). All of the 63 cfDNA samples and tumour tissues had previously been genotyped for EGFR, KRAS, NRAS and BRAF mutations using a TDA (Gonzalez-Cao et al, 2015 (link); Karachaliou et al, 2015 (link)). In the case of tumour tissues, genotyping had been confirmed by standard PCR followed by Sanger sequencing. Cases showing discordance between the NGS SiRe panel and the TDA were further investigated by digital PCR (dPCR) on a QuantStudio 3D Digital PCR System platform (Thermofisher) as previously described (Malapelle et al, 2016b (link)).
5
Digital PCR Protocol for BRAF and KRAS Mutation Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Digital PCR experiment set up was performed according to the Digital MIQE Guidelines37 .
Amplification was carried out in 15 µl volume using the QuantStudio 3D Digital PCR System Platform (Thermo Fisher Scientific, Carlsbad, CA, USA). PCR reaction mix contained 8 µl of 2X QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific), 0.4 µl of 40X TaqMan-MGB-FAM-probe assay (BRAF assay ID: BRAF_476_mu; KRAS G12S Assay ID: Hs000000048; KRAS G13D assay ID: Hs000000056_rm; Thermo Fisher) 1.1 µl of diluted DNA (50 ng/µl) and 6.5 µl of nuclease-free water. Serial dilutions of genomic DNA standards (Cat Num. HD238, Horizon Discovery) and of negative controls with DNA-free water were used to estimate the limit of detection. (Suppl. Fig.3 ).
To amplify the target genes, 15 µl of the reaction mix were loaded onto a QuantStudio 3D Digital PCR 20 K Chip (Thermo Fisher Scientific) and run following this PCR program: 95 °C for 8′, 40 cycles at 95 °C for 15″ and 60 °C for 1′, followed by a final extension step at 60 °C for 2′. Raw data were analyzed with QuantStudio 3D Analysis Suite Cloud Software to calculate gene copies per ml.
Amplification was carried out in 15 µl volume using the QuantStudio 3D Digital PCR System Platform (Thermo Fisher Scientific, Carlsbad, CA, USA). PCR reaction mix contained 8 µl of 2X QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific), 0.4 µl of 40X TaqMan-MGB-FAM-probe assay (BRAF assay ID: BRAF_476_mu; KRAS G12S Assay ID: Hs000000048; KRAS G13D assay ID: Hs000000056_rm; Thermo Fisher) 1.1 µl of diluted DNA (50 ng/µl) and 6.5 µl of nuclease-free water. Serial dilutions of genomic DNA standards (Cat Num. HD238, Horizon Discovery) and of negative controls with DNA-free water were used to estimate the limit of detection. (Suppl. Fig.
To amplify the target genes, 15 µl of the reaction mix were loaded onto a QuantStudio 3D Digital PCR 20 K Chip (Thermo Fisher Scientific) and run following this PCR program: 95 °C for 8′, 40 cycles at 95 °C for 15″ and 60 °C for 1′, followed by a final extension step at 60 °C for 2′. Raw data were analyzed with QuantStudio 3D Analysis Suite Cloud Software to calculate gene copies per ml.
6
Digital PCR Analysis of Tumor Mutations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!