Journal: PLoS Genetics

Article Title: Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA

doi: 10.1371/journal.pgen.1006824

Figure Lengend Snippet: (A) Model of alternative splicing in the 65K gene. Binding of U11/U12 di-snRNP to USSE activates alternative splicing to form an isoform with a long 3′UTR. Exons are in blue and the 3′UTR in yellow. (B) Sashimi plots of RNAseq data in the 3′ end of the Rnpc3 locus ( U11/U12-65K ) gene from mouse stem cells (ESC: embryonic stem cells, NSC: neural stem cells), radial glia-type (RG) and glutamatergic neurons (GN) of increasing maturity (DIV1-28). Junction reads are plotted as arcs with the number indicating number of exon-spanning reads. (C) Sashimi plot of RNAseq reads in region downstream of the Rnpc3 ( U11/U12-65K ) gene and in the Amy1 gene. (D) RT-PCR analysis of Rnpc3 ( 65K ) isoform ratio and Rnpc3-Amy1 conjoined transcripts (upper section) and Western blot analysis (lower section) in mouse cortical neural stem cells and cortical neurons cells. The identities of the amplicons are indicated on left with arrows showing the location of primers. Immunoprecipitation followed by Western blots were performed using a U11/U12 65K antibody (Proteintech, 25820-1-AP) and shown in the panel labeled as "IP". For normalization, p44/42 MAPK (Erk1/2) (Cell Signaling, #4695) were probed in the panel labeled "Load" that represents the sample prior IP. Asterisk indicates the IgG heavy chain. (E) RT-PCR quantification of the indicated isoforms from samples shown in (D). Error bars represent the standard deviation of 3 biological replicates and the asterisk indicates p-value < 0.05 in two-tailed Student′s t-test in (E), (F) and (G). (F) Fold-change quantification by RT-PCR of conjoined Rnpc3-Amy1 transcripts from samples shown in (D). Samples were normalized with total 65K ( Rnpc3) expression. (G) Quantification of the relative U11/U12-65K protein levels from Western blot samples shown in (D). Samples were normalized with p44 MAPK (Erk1) expression. (H) Sashimi plot of ENCODE RNAseq reads in the RNPC3 ( U11/U12-65K ) gene and AMY2B gene from human cerebellum and HeLa S3 cells. Junction reads are plotted as arcs with the number indicating number of reads. Arrows indicate position of USSE, 5’ss, pA (poly(A) site) and IE (intergenic exon).

Article Snippet: Briefly, anti-RNPC3 antibody (Proteintech 25820-1-AP) was added and incubated with rotation at 4°C for 3 hrs.

Techniques: Alternative Splicing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Labeling, Standard Deviation, Two Tailed Test, Expressing

Journal: Microbiology Spectrum

Article Title: Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2

doi: 10.1128/Spectrum.00672-21

Figure Lengend Snippet: Performance of in-house rNP plant- and E. coli -based, SD Biosensor total Ab, and EDI novel coronavirus COVID-19 IgG and IgM ELISAs ^a

Article Snippet: The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Microbiology Spectrum

Article Title: Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2

doi: 10.1128/Spectrum.00672-21

Figure Lengend Snippet: Optical density at 450 nm (OD 450 ) for antibody detection by days after symptom onset. Plant-based rNP (A), E. coli -based rNP (B), SD Biosensor total Ab (C), EDI-IgG (D), and EDI-IgM (E) ELISAs. The gray line shows the calculated cutoff values by mean + 3 SD or range recommended by commercial assay. The red line shows the cutoff values recommended by the ROC curve. Green lines indicate the median with interquartile ranges. The data set contained 286 samples for rNP plant-based, rNP E. coli -based, and SD Biosensor total Ab ELISAs, including 128 samples from 30 symptomatic COVID-19 patients and 158 samples from negative controls. For the EDI-IgG and -IgM ELISAs, 64 samples were analyzed, including 44 samples from 19 symptomatic COVID-19 patients and 20 samples from negative controls. mean + 3 SD, mean optical density plus 3-fold of standard deviation. ROC, receiver operating characteristic.

Article Snippet: The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs.

Techniques: Standard Deviation

Journal: Microbiology Spectrum

Article Title: Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2

doi: 10.1128/Spectrum.00672-21

Figure Lengend Snippet: Sensitivity of in-house rNP plant-based, E. coli -based, SD Biosensor total Ab, and EDI novel coronavirus COVID-19 IgG and IgM ELISAs with the duration of illness ^a

Article Snippet: The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs.

Techniques:

Journal: Microbiology Spectrum

Article Title: Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2

doi: 10.1128/Spectrum.00672-21

Figure Lengend Snippet: Graph of the positive rate of SARS-CoV-2-specific total antibodies, IgG, and IgM by days post-symptom onset. (A) Positive rate obtained from recommended cutoff values by ROC curve. (B) Positive rate obtained from calculated cutoff values by mean + 3 SD or range recommended by commercial assay. The data set contains 128 samples for rNP plant-based and rNP E. coli -based assays from 30 symptomatic COVID-19 patients. For EDI-IgG ELISA, 44 samples were analyzed from 19 symptomatic COVID-19 patients. The cutoffs recommended by ROC curve were 0.5, 1.8, 0.18, 0.28, and 0.09 for rNP plant-based, rNP E. coli -based, SD Biosensor total Ab, EDI-IgG, and EDI-IgM ELISAs, respectively. The calculated cutoff values by mean + 3 SD or range/value recommended by commercial assay were 0.7, 2.2, 0.36, 0.32 to 0.39, and 0.18 to 0.22 for rNP plant-based, rNP E. coli - based, SD Biosensor test Ab, EDI-IgG, and EDI-IgM ELISAs, respectively. The borderline results obtained from the EDI commercial assay were considered positive.

Article Snippet: The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Microbiology Spectrum

Article Title: Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2

doi: 10.1128/Spectrum.00672-21

Figure Lengend Snippet: Receiver operating characteristic (ROC) curves for the evaluation and comparison of diagnostic accuracy. (A) Plant rNP-based, E. coli rNP-based, and SD Biosensor total Ab ELISAs. (B) Plant rNP-based, E. coli rNP-based, SD Biosensor total Ab, EDI-IgG, and EDI-IgM ELISAs. The data set contain all 299 samples for rNP plant-based, rNP E. coli -based, and SD Biosensor total Ab ELISAs, including 141 samples from 32 patients with COVID-19 and 158 samples from negative controls. For comparison with EDI-IgG and -IgM ELISA, the data set contains 71 samples, including 51 samples from 21 patients with COVID-19 and 20 samples from negative controls.

Article Snippet: The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs.

Techniques: Diagnostic Assay, Enzyme-linked Immunosorbent Assay

Journal: Microbiology Spectrum

Article Title: Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2

doi: 10.1128/Spectrum.00672-21

Figure Lengend Snippet: Correlation analysis of the OD values between in-house rNP plant-based, E. coli -based, SD Biosensor total Ab, and EDI novel coronavirus COVID-19 IgG and IgM ELISAs

Article Snippet: The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs.

Techniques: Enzyme-linked Immunosorbent Assay