primers probes ddpcr reference housekeeping gene (rpp30  (Bio-Rad)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Digital droplet PCR analysis of organoids generated from mouse mammary tumors demonstrates proof-of-concept capture of tumor heterogeneity

doi: 10.3389/fcell.2024.1358583

Figure Lengend Snippet: Organoid generation retains clinically relevant tumor genomic heterogeneity. (A) Copy number amplified genes in IDC patients found in distant organ metastasis, unspecified metastasis site, or lymph node metastasis compared to primary breast cancer samples in the GENIE database. Of the genes (dots) amplified in greater than 10% of primary or metastatic samples, the top 10 statistically significant (blue dots) differentially amplified in metastatic samples are circled. These include ADGRA2, RAD21, PAK1, FGF4, NSD3, FGF19, FGF3, CCND1, FGFR1, and MYC. (B) Schema of workflow for sample generation. Large primary mammary tumors are dissected from the fat pad. Tissue segments are excised from 4 distinct regions of the tumor to ensure adequate sampling. Organoids generated from the tumors were sampled and dissociated and strained to single cells, taken as pooled organoid samples, or isolated to single organoids. All samples were then used to isolate gDNA and perform ddPCR. (C) FGFR1 copy numbers in tissue, pooled organoids (Org), and single cell digests (Digest) from two different mice normalized to the housekeeping gene RPP30. Tissue sample copy number alterations are statistically different than pooled organoid samples (Mann-Whitney, p < 0.05). Each point represents separate batches of organoids generated from primary tumor ( n = 2, r = 2). Each primary tumor was split into 4 samples for separate organoid generation to ensure adequate sampling over the whole tumor. (D) FGFR1 copy numbers in single organoids from tumors from two different mice. Among each tumor there exist statistically distinct copy numbers (Kruskall-Wallis, p = 0.0222; p = 0.0032).

Article Snippet: Primers and probes for ddPCR for reference housekeeping gene (RPP30) and target genes (ADGRA2, FGFR1, NSD3, PAK1) were purchased from Bio-Rad Laboratories (Assay IDs: dMmuCNS822293939, dMmuCNS263266645, dMmuCNS890129559, dMmuCNS681547140, dMmuCNS429051281, respectively).

Techniques: Amplification, Sampling, Generated, Isolation, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: Digital droplet PCR analysis of organoids generated from mouse mammary tumors demonstrates proof-of-concept capture of tumor heterogeneity

doi: 10.3389/fcell.2024.1358583

Figure Lengend Snippet: FGFR1 is copy number amplified in both human metastasis and in vitro model of metastasis. (A) Representative image of a non-invading, control group organoid in liquid media (top). Representative image of an invading, experimental group organoid post-collagenase digestion in liquid media (bottom). Both of these organoids were initially allowed to grow either in media (control) or type-1 collagen ECM (experimental) for 48 h. These images are representative of the conditions assessed in this figure (B) . (B) FGFR1 copy number is amplified in invading organoids vs. control organoids from PyMT mouse primary mammary tumor samples (Mann-Whitney, p = 0.0022). Each point represents copy number measured from duplicate control or invasive organoid samples from 2 PyMT mouse primary mammary tumor organoids. Duplicates were performed for each sample in ddPCR. Duplicates were not included for those >20% apart.

Article Snippet: Primers and probes for ddPCR for reference housekeeping gene (RPP30) and target genes (ADGRA2, FGFR1, NSD3, PAK1) were purchased from Bio-Rad Laboratories (Assay IDs: dMmuCNS822293939, dMmuCNS263266645, dMmuCNS890129559, dMmuCNS681547140, dMmuCNS429051281, respectively).

Techniques: Amplification, In Vitro, MANN-WHITNEY