Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Lz expansion is associated with increasing levels of circulating and endothelial IGF2 (A) Weights of micro-dissected Lz. (B) Linear correlation analyses between fetal and Lz weights: p = 0.002 (E14), p < 0.0001 (E16), and p < 0.0001 (E19) (n = 46–189 placentae from n > 10 L per group in [A] and [B]). (C) Levels of IGF2 (ng/mL) in plasma of wild-type fetuses. (D) Linear correlation analyses between fetal weights and circulating IGF2: p < 0.0001 (E16 and E19) (n = 70–79 per group in [C] and [D]). (E) Igf2 mRNA in situ hybridization (blue) in E14 wild-type Lz (red arrows—FPEC, feto-placental endothelial cells; AS, antisense probe; inset with S, sense probe; scale bars, 20 μm). (F) Relative Igf2 mRNA expression levels measured by qRT-PCR in FPEC from wild-type Lz (n = 6–7 per group). (G) Imprinted genes that rank within top 100 expressed genes in E16 wild-type FPEC (FPKM, fragments per kilobase million; n = 4). (H) Double immunostaining for IGF2 and CD31 in E19 wild-type placenta. Endothelial cells are very thin and hard to detect except where the cytoplasm is more voluminous around the nucleus, with intense IGF2 stain (white arrows). Transmembrane glycoprotein CD31 immunostaining is in the membrane and largely marks endothelial intercellular junctions (scale bars, 20 μm). (I) Semi-quantitative measurement of IGF2 protein in FPEC versus trophoblast cells (E19 wild-type Lz, n = 60 cells per group from two placentae). White arrows—endothelial cells; scale bars, 50 μm. For (E), (H), and (I): FC, fetal capillaries; MBS, maternal blood spaces; LT, labyrinthine trophoblast cells; S-TGC, sinusoidal trophoblast giant cells. Data in (A), (C), (F), (G), and (I) are presented as averages ± standard deviation (SD); ∗∗∗ p < 0.001 calculated by one-way ANOVA plus Tukey’s multiple comparisons test in (A) and (F) or by unpaired t test with Welch’s correction in (C) and (I). See also .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Clinical Proteomics, In Situ Hybridization, Expressing, Quantitative RT-PCR, Double Immunostaining, Staining, Immunostaining, Membrane, Standard Deviation

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Deletion of Igf2 in the epiblast or endothelium impairs Lz expansion (A) Left: schematic of Igf2 expression in conceptuses with conditional deletion driven by Meox2 Cre . Right: immunostaining for YFP (green) in a representative fetus and placenta paraffin section at E12 of gestation, double transgenic for Meox2 Cre and Rosa26 fl STOP fl YFP 10 reporter. YFP expression in the placenta is localized to the Lz and Cp (high magnification, inset). Blue—DAPI stain for nuclei; scale bars: 1 mm (low magnification) and 100 μm (high magnification). (B) Fetal and placental growth kinetics, measured as average wet-weights for each genotype per litter (E12: n = 10 L [n = 41 controls {C} and n = 32 Igf2 EpiKO ]; E14: n = 25 L [n = 114 C and n = 88 Igf2 EpiKO ]; E16: n = 37 L [n = 154 C and n = 127 Igf2 EpiKO ]; E19: n = 37 L [n = 164 C and n = 121 Igf2 EpiKO ]). (C) Absolute volumes of the placental layers (Db, decidua basalis; Jz, junctional zone; Lz, labyrinthine zone; Cp, chorionic plate), measured by stereology (n = 6 per group). (D) Absolute volumes of Lz components, measured by stereology (LT, labyrinthine trophoblast; MBSs, maternal blood spaces; FCs, fetal capillaries) (n = 6 per group). (E) Left: schematic representation of Igf2 expression in conceptuses with conditional deletion driven by Tek Cre . Right: representative confocal microscopy of frozen sections from a fetus and its corresponding placenta, double transgenic for TeK Cre and Ai9(RCL-tdT) reporter at E16 of gestation. Scale bars: 2 mm (fetus) and 1 mm (placenta). (F) Fetal and placental growth kinetics (E12: n = 5 L [n = 17 C and n = 16 Igf2 ECKO ]; E14: n = 8 L [n = 26 C and n = 34 Igf2 ECKO ]; E16: n = 13 L [n = 60 C and n = 46 Igf2 ECKO ]; E19: n = 7 L [n = 31 C and n = 27 Igf2 ECKO ]). (G) Absolute volumes of the placental layers measured by stereology (n = 5–7 per group). (H) Absolute volumes of Lz components, measured by stereology (n = 5–7 per group). (I) Double immunostaining for EPCAM (epithelial cell adhesion molecule) (red) and MCT1 (monocarboxylate transporter 1) (green) in a representative frozen placental section at E12 of gestation. EPCAM expression is observed as clusters of positive cells within the Lz placenta. Blue—DAPI (4′,6-diamidino-2-phenylindole) stain for nuclei; scale bars: 500 μm (left panel) and 20 μm (right panel). (J) Analysis of EPCAM high -positive cells by flow cytometry. Left panel: example of gating used to identify EPCAM high -positive cells (the viability dye 7-aminoactinomycin D [7-AAD] was used to exclude dead cells). Right: quantification of placental EPCAM high -positive cells at E12 in conceptuses with conditional Igf2 deletion driven by Meox2 Cre (n = 10 C and n = 9 Igf2 EpiKO from 2 L) or Tek Cre (n = 8 C and n = 8 Igf2 ECKO from 2 L). For all graphs data are shown as averages; error bars represent SD in (C), (D), (G), (H), and (J) or 95% confidence intervals (95% CI) in (B) and (F); N.S.—statistically not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by a mixed effects model in (B) and (F) (see STAR Methods), two-way ANOVA plus Sidak’s multiple comparisons tests in (D) and (H) or unpaired t tests in (C), (G), and (J). See also .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Expressing, Immunostaining, Paraffin Section, Transgenic Assay, Staining, Confocal Microscopy, Double Immunostaining, Flow Cytometry

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Lack of fetus-derived IGF2 reduces the expansion of feto-placental microvasculature in late gestation (A) Functions enriched in DEGs at E19. (B) qRT-PCR analysis of angiopoietin-Tie2/TEK signaling components in Lz (n = 6–8 per group). (C) TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in E16 Lz (arrows point to apoptotic cells) and data quantification (n = 6 samples per group); scale bars, 50 μm. (D) Left: representative double immunostaining for TUNEL (red) and laminin (green, marker of feto-placental capillaries) in the Lz of an E16 Igf2 EpiKO mutant placenta (DAPI, blue marks the nuclei; white and red arrows indicate TUNEL + FPECs and LT, respectively; scale bars, 25 μm). Right: quantification of TUNEL + cells that are positive or negative for laminin (n = 6 Igf2 EpiKO mutant placentae). (E) Feto-placental endothelial cell (FPEC) proliferation measured by flow cytometry (left—representative histograms at E16; right—data quantification; n = 4–11 per group). (F) qRT-PCR analysis of Adgre1 in Lz. (G) Representative F4/80 immunostainings in E16 Lz (arrows indicate macrophages). Scale bars, 100 μm. Right: percentage of macrophages/Lz at E16 (n = 6–8 samples per group). (H) Representative CD31 immunostaining in Lz (scale bars, 100 μm). (I) qRT-PCR analysis for SynT-II (syncytiotrophoblast layer II) marker genes. For all graphs, data are presented as averages or individual values; error bars are SD; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by two-way ANOVA plus Sidak’s multiple comparisons tests in (B), (C), (E), (F), and (I) or Mann-Whitney tests in (G). See also Figure S4 and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Derivative Assay, Quantitative RT-PCR, TUNEL Assay, Staining, Double Immunostaining, Marker, Mutagenesis, Flow Cytometry, Immunostaining, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: Genetic models of mismatched placental and fetal growth reveal circulating IGF2 as a major endocrine regulator of FPEC and Lz expansion (A–E) Column 1: schematic diagrams of the genetic models: Igf2 EpiKO (A), Igf2 ECKO (B), Igf2 TrKO (C), Igf2 UbKO (D), and H19 -DMD EpiKO (E). Columns 2 and 3: total numbers (column 2) and proportion of FPEC/Lz (column 3), measured by flow cytometry (n conceptuses per group: Igf2 EpiKO : n = 9–18; Igf2 ECKO : n = 5–11; Igf2 TrKO : n = 6–17; Igf2 UbKO : n = 3–26; H19 -DMD EpiKO : n = 9–15). Column 4: Lz growth kinetics ( Igf2 EpiKO : n = 9–20 L; Igf2 ECKO : n = 3–9 L; Igf2 TrKO : n = 4–9 L; Igf2 UbKO : n = 3–8 L; H19 -DMD EpiKO : n = 3–4 L). Column 5: IGF2 levels (ng/mL) in plasma (n per group: Igf2 EpiKO : n = 12; Igf2 ECKO : n = 9; Igf2 TrKO : n = 6–7; Igf2 UbKO : n = 7–11; H19 -DMD EpiKO : n = 9). Data are shown as averages or individual values and error bars are SD (columns 2, 3, and 5) and 95% CI (column 4). N.S.—not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by two-way ANOVA plus Sidak’s multiple comparisons tests (second and third columns), mixed effects model (fourth column) or Mann-Whitney tests (fifth column). See also A, S5B, and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Flow Cytometry, Clinical Proteomics, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 signaling regulates angiogenic properties of endothelial cells (A) Volcano plot representation of DEGs identified by RNA-seq in E16 FPEC ( Igf2 EpiKO versus controls). Significant upregulated and downregulated DEGs (false discovery rate [FDR] < 0.05) are shown with red and blue, respectively. (B) Top scoring biological processes enriched in DEGs. Biologically validated DEGS are listed in parentheses. The dotted line corresponds to FDR-corrected p value of 0.05. (C) Biological validation. Data are shown as averages (n = 11–12 samples per group); error bars are SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 calculated by Mann-Whitney tests. (D) Volcano plot representation of DEGs identified by RNA-seq in E16 FPEC ( Igf2 ECKO versus controls). Significant upregulated and downregulated DEGs (FDR < 0.05) are shown with red and blue, respectively. (E) Transcription factors (TFs) identified by analysis of motif enrichment (AME). (F) IPA regulatory network built with the four TFs identified using AME analysis. Proteins labeled with a star are known regulators of angiogenesis (angiostatic or pro-angiogenic factors) and key references are listed in . See also C and S5D and and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: RNA Sequencing, Biomarker Discovery, MANN-WHITNEY, Labeling

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 Acts on FPECs via IGF2R-ERK signaling ex vivo (A) Primary FPEC isolated from E16 Lz: D0—freshly isolated cells; D10—FPEC at passage one (P1, 10 days of culture). (B) Confocal imaging of passage one FPEC, stained for CD31 (scale bars, 20 μm). (C) Flow cytometry analysis of P1 FPEC stained for CD31, demonstrating that these are almost exclusively CD31 + . (D) qRT-PCR analysis for Igf1r , Igf2r , and Insr in FPECs isolated by FACS (n = 6–7 per group). (E) Relative expression of the three IGF receptors in P1 FPEC. (F) qRT-PCR analysis of Igf2 mRNA levels in P1 FPEC cultured in 5% O 2 versus primary FPEC isolated from E16 Lz by FACS. (G) Schematic representation of IGF2 and IGF receptors. IGF2 Leu27 analog acts specifically on IGF2R and picropodophyllin (PPP) inhibits phosphorylation of IGF1R. (H) Representative images of capillary-like tube formation assay in primary FPEC seeded on matrigel and exposed to exogenous IGF2, IGF2 Leu27 , PPP, or PPP+IGF2 (equal seeding of cell numbers at 30 min and tube formation at 8 h), and quantification of number of nodes, branches, and total length (n = 5–6 independent experiments). (I) qRT-PCR analysis of Igf2r mRNA levels in primary FPECs upon knockdown by siRNA (n = 8 samples/group). (J) Proliferation assay of primary FPEC with or without IGF2R siRNA knockdown, in presence or absence of IGF2, on 4 consecutive days after plating. Cells with IGF2R siRNA knockdown exhibit significant proliferation defects that are further accentuated upon IGF2 treatment (n = 5 biological replicates per group). (K) qRT-PCR analysis of Angpt2 mRNA levels in primary FPECs transfected with scrambled siRNA or IGF2R siRNA, upon 4 days of treatment with 50 ng/mL mouse recombinant IGF2 (n = 8 samples/group). (L) Left side: identification of delayed ERK1/2 phosphorylation in FPECs with IGF2R siRNA knockdown upon acute treatment with 50 ng/mL mouse recombinant IGF2. HSP90 was used as internal control for protein loading. Right side: quantification of ratios pERK1/2 to total ERK1/2 for n = 3 independent biological replicates. For all graphs, data are presented as averages or individual values and error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 calculated by a Mann-Whitney test in (F), two-way ANOVA tests with Sidak’s multiple comparisons test in (H), (J), and (L), Wilcoxon matched-pairs signed rank test in (I) and paired Student’s t test in (K).

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Ex Vivo, Isolation, Imaging, Staining, Flow Cytometry, Quantitative RT-PCR, Expressing, Cell Culture, Phospho-proteomics, Capillary Tube Formation Assay, Knockdown, Proliferation Assay, Transfection, Recombinant, Control, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet: IGF2 acts on FPECs via IGF2R in vivo (A) Representative double immunostaining for IGF2R (red) and CD31 (green) in Igf2r ECKO mutant and control Lz at E16 (DAPI, blue; scale bars, 25 μm). (B) Flow cytometry analysis showing that the majority (>80%) of Igf2r ECKO mutant feto-placental endothelial cells (FPECs) express YFP (n = 6–14 per genotype). (C) Fetal and placental growth kinetics in Igf2r ECKO ( Igf2r fl/+ ; Tek +/Cre ) mutants compared with Igf2r fl/+ controls (n = 8–28 conceptuses from n = 3–8 L for each developmental stage). (D) Proportion and total numbers of FPEC/Lz measured by flow cytometry (n = 6–14 per group). (E) Representative CD31 staining in E16 Lz (scale bars, 100 μm). (F) Lz growth kinetics: Igf2r ECKO (n = 8–16 conceptuses per group). (G) IGF2 levels (ng/mL) in plasma at E16 (n = 9 per group). (H) Model summarizing the proposed actions of fetus-, endothelial-, and trophoblast-derived IGF2. For all graphs, data are presented as averages or individual values and error bars represent SD in (B), (D), and (G), or 95% CI in (C) and (F). N.S.— not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 calculated by two-way ANOVA tests in (B) and (D), mixed effects model in (C) and (F) and Mann-Whitney tests in (G). See also Figure S7 and .

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: In Vivo, Double Immunostaining, Mutagenesis, Control, Flow Cytometry, Staining, Clinical Proteomics, Derivative Assay, MANN-WHITNEY

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet:

Article Snippet: Sub-confluent cells (∼80%) at passage one (around 10 days in culture) were washed and then cultured in 5% serum replacement media (Sigma – S0638) for ∼40 h. From each litter we used cells at passage one for treatment with 50 ng/ml mouse recombinant IGF2 (R&D Systems, 792-MG-050; dissolved in PBS), 1000 ng/ml human IGF2 Leu27 (GroPep – TU100; dissolved in 10mM HCl), 500nM picropodophyllotoxin (PPP, Sigma – T9576; dissolved in DMSO) or 500nM PPP + 50 ng/ml IGF2, or appropriate vehicle control.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, BIA-KA, Western Blot, Stripping Membranes, Reverse Transcription, SYBR Green Assay, Red Blood Cell Lysis, Staining, Enzyme-linked Immunosorbent Assay, In Situ, TUNEL Assay, Imaging, Flow Cytometry, Transfection, Gene Expression, Expressing, Microarray, Isolation, Software

Journal: Sensors (Basel, Switzerland)

Article Title: COVID-19 Testing and Diagnostics: A Review of Commercialized Technologies for Cost, Convenience and Quality of Tests

doi: 10.3390/s21196581

Figure Lengend Snippet: At-Home Molecular Diagnostic (RT-PCR) Tests using Respiratory Specimens and granted FDA Emergency Use Authorization.

Article Snippet: Bio-Rad Laboratories , Bio-Rad SARS-CoV-2 ddPCR Test (05/01/2020) , Reverese transcriptase droplet digital PCR test, for prescription use only , Supermix, reverse transcriptase (RT), and 300 mM dithiothreitol (DTT) solution.

Techniques: Diagnostic Assay, Microarray, Hybridization, Digital PCR, Quantitative RT-PCR, Mass Spectrometry, Amplification, Lysis, Negative Control, Positive Control

Journal: Sensors (Basel, Switzerland)

Article Title: COVID-19 Testing and Diagnostics: A Review of Commercialized Technologies for Cost, Convenience and Quality of Tests

doi: 10.3390/s21196581

Figure Lengend Snippet: At-Home Molecular Diagnostic (RT-PCR) Tests using Saliva Specimens and granted FDA Emergency Use Authorization.

Article Snippet: Bio-Rad Laboratories , Bio-Rad SARS-CoV-2 ddPCR Test (05/01/2020) , Reverese transcriptase droplet digital PCR test, for prescription use only , Supermix, reverse transcriptase (RT), and 300 mM dithiothreitol (DTT) solution.

Techniques: Diagnostic Assay, Quantitative RT-PCR, Digital PCR, Labeling, High Throughput Screening Assay, Transferring

Journal: Emerging Infectious Diseases

Article Title: Identifying Influenza Viruses with Resequencing Microarrays

doi: 10.3201/eid1204.051441

Figure Lengend Snippet: Hybridization images of the respiratory pathogen microarray (RPM) version 1 prototype regions for 3 influenza virus isolates and trivalent FluMist vaccine. A) A/H1N1, B) A/H3N2, C) influenza B, and D) trivalent FluMist vaccine. In A, B, and C, only the influenza-specific tiled prototype regions of RPM version 1 are shown. Hybridization-positive identifications are shown on the right. In D, the image of the entire RPM version when hybridized with FluMist vaccine is shown. The single influenza prototype region that was hybridization negative is denoted on the right. E) Magnification of a portion of profile B showing an example of the primary sequence data generated by the hybridization of randomly amplified targets to the RPM version 1 HA3 probe set. The primary sequence generated can be read from left to right. HA, hemagglutinin; NA, neuraminidase; IQEX, internal positive hybridization control (Affymetrix); M, matrix.

Article Snippet: After the prehybridization step, 167.5 μL of hybridization cocktail master mix (3 mol/L tetramethylammonium chloride, 10 mmol/L Tris, pH 7.8, 0.01% Tween 20, 0.5 mg/mL bovine serum albumin, 0.1 mg/mL herring sperm DNA [Promega, Madison, WI, USA], 50 pmol/L Oligo B2 [Affymetrix Inc.]) and biotin-labeled DNA fragments were heated for 5 min at 95°C, equilibrated for 5 min at 45°C, and added to RPM version 1.

Techniques: Hybridization, Microarray, Virus, Sequencing, Generated, Amplification, Control