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Image Search Results

Journal: Molecular and cellular endocrinology
Article Title: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription
doi: 10.1016/j.mce.2016.04.001
Figure Lengend Snippet: Improved q-PCR quantitation of StAR pre-mRNA (p-RNA). (A) PCR primer pairs that target the primary transcripts (E1/I1; E6/I6) spliced transcripts (E1/E2; E3/E4; E5/E6) and 30UTR (E7-S (short) and E7-L (long)). (B) The composite gBlock used as an RT-qPCR quantitation standard, which includes all the target intron, exon and 30UTR sequences of StAR gene synthesized on the same genomic block. (C) The time course of stimulation of p-RNA (E1/I1) and mRNA (E5/E6) in MA10 cells with Br-cAMP. (D) RT priming is affected by the location of the specific RT primers at location E1/I1 (S is the reverse primer of E1/I1) relative to the qPCR 30 site (a). The bar graph shows the relationship between the copy number and the priming distance. (b). Line graph (in box) shows the relationship between the free energy and the priming distance (c). (E) RT priming effect is again similarly observed in E6/I6 of StAR gene. The display is a repeat of 1 D (a–c). (F) Comparison of the predicted lowest free energy structures. Data in (C–E) are given as mean ± SEM. p < 0.05, **p < 0.01, ***p < 0.001 by student’s t-test or ANOVA. (CeE) 1 mM of Br-cAMP stimulation in MA-10 cells.
Article Snippet:
Techniques: Quantitation Assay, Quantitative RT-PCR, Synthesized, Blocking Assay

Journal: Molecular and cellular endocrinology
Article Title: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription
doi: 10.1016/j.mce.2016.04.001
Figure Lengend Snippet: Application of optimized q-PCR to stimulation of StAR p-RNA and sp-RNA. (A) Spliced transcripts formed with the random hexamers, oligo-dT, and specific 3’qPCR primer are compared. (B) The display is a repeat of C with Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) primer set. (C) Comparison of E1/I1 assessment with only the individual specific RT primers or with a cocktail of RT primers of multiple StAR targets. (D) The time course of stimulation of p-RNA (E1/I1) and mRNA (E5/E6) using DNA block references to provide transcript copy number. Data in (A–D) are given as mean ± SEM. p < 0.05, **p < 0.01, ***p < 0.001 by student’s t-test or ANOVA. (AeD) 1 mM of Br-cAMP stimulation in MA-10 cells.
Article Snippet:
Techniques: Blocking Assay

Journal: Molecular and cellular endocrinology
Article Title: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription
doi: 10.1016/j.mce.2016.04.001
Figure Lengend Snippet: Time course for stimulation of 3’UTR transcripts in comparison to StAR transcripts (p-RNA, sp-RNA). (A) Comparison of spliced transcripts formed at three different spliced sites (E1/E2; E3/E4 and E5/E6) as a function of time of stimulation. (B) Comparison of spliced transcripts formed at E5/E6 and 30 UTR (E7-S and E7-L). (C) 30’UTR (E7-S and E7-L) are compared with the primary transcripts (E1/I1 and E6/I6). (D) The display is a repeat of 1 C with a different batch of lower passage MA10 cells. Data in (A-D) are given as mean ± SEM. p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA. (A-D) 1 mM of Br-cAMP stimulation in MA-10 cells.
Article Snippet:
Techniques:

Journal: Molecular and cellular endocrinology
Article Title: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription
doi: 10.1016/j.mce.2016.04.001
Figure Lengend Snippet: HR-FISH analysis of StAR RNA distribution in MA10 cells. (A) The design of HR-FISH probes that distinguish StAR p-RNA, sp-RNA and the extended 3’UTR of the 3.5 kb mRNA. (B) Overlap (yellow) of p-RNA (green) with sp-RNA (red) fluorescence signal at the loci at 60 min (C) StAR mRNAs accumulate in the cytoplasm after 180 min. (D) Equivalent Z-projection image using both sp-RNA probe set and the 3’EU probe set to detect StAR mRNA in the cytoplasm. White specks in the merged images represent dual binding of 3.5 kb mRNA. (E) Z-projection merger of sp-RNA/mRNA with StAR protein, which colocalizes within all mitochondria (Mitotracker) (not shown). (F) The merger of the sp-RNA (red) and 3’EU (green) (overlap yellow) at nuclear loci (1,2). StAR loci position around the nuclear midline in Z-stack series XY slices (step size: 0.2 μm). The diagram represents positions for XY slices relative to the nucleus. (C–F) 1 mM of Br-cAMP stimulation in MA-10 cells for 3 h s, (B) for 1hr. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Article Snippet:
Techniques: Fluorescence, Binding Assay

Journal: Molecular and cellular endocrinology
Article Title: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription
doi: 10.1016/j.mce.2016.04.001
Figure Lengend Snippet: Resolution of StAR 3.5 kb mRNA by dual binding of sp-RNA and 3’EU, and pairing of StAR mRNA and mitochondria (N-SIM microscope). (A) 3-D resolution of StAR sp-RNA/mRNA and mitochondria marked by StAR protein immunofluorescence. (B) Z-slice resolution for StAR sp-RNA at Z axis XY slice positions a, b and c (see Fig. 4F). Above: Diagram showing separation of matrix StAR protein from StAR mRNA position with ribosomes linked to the mitochondrial outer membrane (OMM). The diameter of adrenal mitochondria is about 500 nm. Lower right: enlargement of a representative sector of the slice (b) showing pairing of StAR protein/mitochondria with StAR mRNA. (C) Z slices showing hybridization of sp-RNA, 3’EU and their merger at the same Z slice positions; a, b, and c. (D) Copies per slice of respectively sp-RNA and 3’EU at positions a, b and c and intermediate slice positions. Shows trend towards the enrichment of p-RNA proximal to the adherent cell membrane. (AeD) 1 mM of Br-cAMP stimulation in MA-10 cells for 3 h s.
Article Snippet:
Techniques: Binding Assay, Microscopy, Immunofluorescence, Hybridization

Journal: Molecular and cellular endocrinology
Article Title: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription
doi: 10.1016/j.mce.2016.04.001
Figure Lengend Snippet: Automatic counting of transcripts in 3D HR-ISH images. (A) mRNAs detected by the spot inspector in the ‘FISH quant’ software (C) Modeling of the sp-RNA conglomeration at the primary locus based on an average single mRNA image (upper panel). sp-RNA quantification algorithm as applied at the locus (lower panel). Compare estimated sp-RNA at StAR loci after 60 min versus 180 min (right panel). (D) The number of sp-RNA (loci plus cytoplasm) from 25 individual cells compared to expression levels (copies per cell) determined by 3’qPCR for six macro-cultures. Data (B–C), Student’s t-test: **, p < 0.01. (A, C) 1 mM of Br-cAMP stimulation in MA-10 cells for 3 h s.
Article Snippet:
Techniques: Software, Expressing