green fluorescent single molecule mrna fish probe  (Thermo Fisher)


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    Thermo Fisher green fluorescent single molecule mrna fish probe
    RNAi depletion of ALPH1 causes growth arrest and increase in <t>mRNA</t> levels. RNAi depletion of TbALPH1 was induced by tetracycline (TET). Three independent clonal cell lines were analysed over a time-course of RNAi induction. (A) Reduction in the number of ALPH1 mRNA molecules. ALPH1 mRNA and DBP1 mRNA (control) were visualized by dual colour single molecule mRNA <t>FISH</t> (Affymetrix), using green ( ALPH1 ) and red ( DBP1 ) fluorescent Affymetrix probe sets. The number of mRNA molecules per cell was quantified after 0, 24 and 48 hours of ALPH1 RNAi induction. Data are presented as box plot (waist is median; box is IQR; whiskers are ±1.5 IQR; only the smallest and largest outliers are shown; n = 100 for each time-point); the number of mRNAs from the individual cells is also presented as green ( ALPH1 ) or red ( DBP1 ) dots. One representative cell for each time-point is shown. The data are from one RNAi clone; data of a second clone are shown in S12 Fig . (B) Growth arrest. Growth was measured over a time-course of ALPH1 RNAi induction (±TET). Averages of the three clonal cell lines are shown; error bars indicate standard deviations between the three cell lines. (C-E) Increase in mRNAs. Total RNA was isolated over a time-course of ALPH1 RNAi and as a control from bloodstream form trypanosomes (BSF) and analysed by northern blots. The blots were probed for total mRNA with an oligo antisense to the miniexon of the spliced leader RNA (C), for PGKC (D) and for GPI-PLC (E). mRNA abundances were quantified by Odyssey (total mRNA) or phosphorimager ( PGKC , GPI-PLC ). rRNA was used as a loading control and all samples were calibrated to the amount of BSF RNA (= 1). Average values of the three clones are shown, standard deviations are presented as error bars. For each probe, one representative northern blot is shown. Note that the three red bands in C) are not rRNA bands, but are caused by a squeezing of the mRNAs due to the very abundant rRNA.
    Green Fluorescent Single Molecule Mrna Fish Probe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes"

    Article Title: The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006456

    RNAi depletion of ALPH1 causes growth arrest and increase in mRNA levels. RNAi depletion of TbALPH1 was induced by tetracycline (TET). Three independent clonal cell lines were analysed over a time-course of RNAi induction. (A) Reduction in the number of ALPH1 mRNA molecules. ALPH1 mRNA and DBP1 mRNA (control) were visualized by dual colour single molecule mRNA FISH (Affymetrix), using green ( ALPH1 ) and red ( DBP1 ) fluorescent Affymetrix probe sets. The number of mRNA molecules per cell was quantified after 0, 24 and 48 hours of ALPH1 RNAi induction. Data are presented as box plot (waist is median; box is IQR; whiskers are ±1.5 IQR; only the smallest and largest outliers are shown; n = 100 for each time-point); the number of mRNAs from the individual cells is also presented as green ( ALPH1 ) or red ( DBP1 ) dots. One representative cell for each time-point is shown. The data are from one RNAi clone; data of a second clone are shown in S12 Fig . (B) Growth arrest. Growth was measured over a time-course of ALPH1 RNAi induction (±TET). Averages of the three clonal cell lines are shown; error bars indicate standard deviations between the three cell lines. (C-E) Increase in mRNAs. Total RNA was isolated over a time-course of ALPH1 RNAi and as a control from bloodstream form trypanosomes (BSF) and analysed by northern blots. The blots were probed for total mRNA with an oligo antisense to the miniexon of the spliced leader RNA (C), for PGKC (D) and for GPI-PLC (E). mRNA abundances were quantified by Odyssey (total mRNA) or phosphorimager ( PGKC , GPI-PLC ). rRNA was used as a loading control and all samples were calibrated to the amount of BSF RNA (= 1). Average values of the three clones are shown, standard deviations are presented as error bars. For each probe, one representative northern blot is shown. Note that the three red bands in C) are not rRNA bands, but are caused by a squeezing of the mRNAs due to the very abundant rRNA.
    Figure Legend Snippet: RNAi depletion of ALPH1 causes growth arrest and increase in mRNA levels. RNAi depletion of TbALPH1 was induced by tetracycline (TET). Three independent clonal cell lines were analysed over a time-course of RNAi induction. (A) Reduction in the number of ALPH1 mRNA molecules. ALPH1 mRNA and DBP1 mRNA (control) were visualized by dual colour single molecule mRNA FISH (Affymetrix), using green ( ALPH1 ) and red ( DBP1 ) fluorescent Affymetrix probe sets. The number of mRNA molecules per cell was quantified after 0, 24 and 48 hours of ALPH1 RNAi induction. Data are presented as box plot (waist is median; box is IQR; whiskers are ±1.5 IQR; only the smallest and largest outliers are shown; n = 100 for each time-point); the number of mRNAs from the individual cells is also presented as green ( ALPH1 ) or red ( DBP1 ) dots. One representative cell for each time-point is shown. The data are from one RNAi clone; data of a second clone are shown in S12 Fig . (B) Growth arrest. Growth was measured over a time-course of ALPH1 RNAi induction (±TET). Averages of the three clonal cell lines are shown; error bars indicate standard deviations between the three cell lines. (C-E) Increase in mRNAs. Total RNA was isolated over a time-course of ALPH1 RNAi and as a control from bloodstream form trypanosomes (BSF) and analysed by northern blots. The blots were probed for total mRNA with an oligo antisense to the miniexon of the spliced leader RNA (C), for PGKC (D) and for GPI-PLC (E). mRNA abundances were quantified by Odyssey (total mRNA) or phosphorimager ( PGKC , GPI-PLC ). rRNA was used as a loading control and all samples were calibrated to the amount of BSF RNA (= 1). Average values of the three clones are shown, standard deviations are presented as error bars. For each probe, one representative northern blot is shown. Note that the three red bands in C) are not rRNA bands, but are caused by a squeezing of the mRNAs due to the very abundant rRNA.

    Techniques Used: Fluorescence In Situ Hybridization, Isolation, Northern Blot, Planar Chromatography, Clone Assay

    RNAi depletion of ALPH1 causes an increase in intact mRNA molecules, but no increase in 5’-3’ decay intermediates. (A) Experimental design: The endogenous very long and short-lived mRNA Tb427.01.1740 was used as a reporter for mRNA metabolism [ 55 ]. Simultaneous probing of the extreme 5’ and 3’ ends with red and green fluorescent single mRNA FISH probes results in yellow, green or red fluorescent mRNA molecules, corresponding to intact mRNAs, mRNAs in 5’-3’ decay and mRNAs in transcription or 3’-5’ decay, respectively. (B) The numbers of yellow, green and red fluorescent spots were quantified from cells after induction of ALPH1 RNAi (0, 24 or 48 hours TET) from at least 130 cells per time-point, for three RNAi clones. Average data are shown; error bars represent the standard deviations between the different RNAi clones. The numbers of the differently coloured spots per total cell are shown (left), but also the numbers of spots in the nucleus (middle) or in the cytoplasm (right). If the centre of a spot was overlapping with the DAPI stained nucleus on a Z-stack projection image, the spot was defined as nuclear, otherwise as cytoplasmic. (C) Representative Z-stack projection images of untreated cells (no TET) and cells after 48 hours of ALPH1 RNAi (48 h TET).
    Figure Legend Snippet: RNAi depletion of ALPH1 causes an increase in intact mRNA molecules, but no increase in 5’-3’ decay intermediates. (A) Experimental design: The endogenous very long and short-lived mRNA Tb427.01.1740 was used as a reporter for mRNA metabolism [ 55 ]. Simultaneous probing of the extreme 5’ and 3’ ends with red and green fluorescent single mRNA FISH probes results in yellow, green or red fluorescent mRNA molecules, corresponding to intact mRNAs, mRNAs in 5’-3’ decay and mRNAs in transcription or 3’-5’ decay, respectively. (B) The numbers of yellow, green and red fluorescent spots were quantified from cells after induction of ALPH1 RNAi (0, 24 or 48 hours TET) from at least 130 cells per time-point, for three RNAi clones. Average data are shown; error bars represent the standard deviations between the different RNAi clones. The numbers of the differently coloured spots per total cell are shown (left), but also the numbers of spots in the nucleus (middle) or in the cytoplasm (right). If the centre of a spot was overlapping with the DAPI stained nucleus on a Z-stack projection image, the spot was defined as nuclear, otherwise as cytoplasmic. (C) Representative Z-stack projection images of untreated cells (no TET) and cells after 48 hours of ALPH1 RNAi (48 h TET).

    Techniques Used: Fluorescence In Situ Hybridization, Clone Assay, Staining

    Related Articles

    Fluorescence In Situ Hybridization:

    Article Title: The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes
    Article Snippet: .. The extreme 5’ and 3’ ends of this mRNA are simultaneously probed with a red and green fluorescent single molecule mRNA FISH probe (Affymetrix), respectively. ..

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    Thermo Fisher zeb2 mrna expression level detection
    Expression of miR-200b-3p, miR-200c-3p, and their downstream target <t>ZEB2</t> <t>mRNA</t> in carcinoma tissues of non-IBC and IBC in comparison with normal breast tissue samples. ( A ) miR-200b-3p and ( B ) miR-200c-3p expression levels are significantly repressed in carcinoma tissues of IBC relative to non-IBC as determined by qPCR, whereas their expression levels in non-IBC and IBC are elevated when compared to normal breast tissue samples. miR-200b-3p and miR-200c-3p expression are log2-transformed and normalized to values of normal tissues collected during reduction mammoplasty. # p
    Zeb2 Mrna Expression Level Detection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher oskar gfp mrna
    Functionality tests of the <t>GFP-tagged</t> fTRG lines by genetic complementation analysis. ( A ) Genetic rescue statistics of null/strong mutant alleles for 46 selected fTRG lines. Note that more than two-thirds of the lines show a rescue (see Table 2 ). ( B , C ) osk -GFP <t>mRNA</t> (in yellow) expressed from fTRG1394 rescues egg-chamber development of an osk null allele ( Jenny et al., 2006 ). osk -GFP mRNA enriches in the early oocyte ( B , stage 6) and rescues the oogenesis arrest and the DNA condensation defect of the osk mutant ( B’ , yellow arrowhead). At stage 10 osk -GFP RNA enriches at the posterior pole ( C ) and produces sufficient protein to ensure proper embryogenesis. osk -GFP mRNA is shown in yellow, DAPI in magenta; scale bars indicate 30 µm. DOI: http://dx.doi.org/10.7554/eLife.12068.006
    Oskar Gfp Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nob1 mrna levels
    RIOK2 and <t>NOB1</t> <t>mRNA</t> expression levels in NSCLC tissues compared with normal tissues. ( a ) The mean RIOK2 mRNA expression level was higher in the tumour tissues (2.16 ± 0.57) than in the corresponding paired adjacent normal lung tissues (0.46 ± 0.21) ( P
    Nob1 Mrna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mrna expression levels
    NRP1 regulates sFlt1 expression and as a consequence blood vessel guidance. ( a – d ) Representative images of ISH for Nrp1 in mouse SC sections at the developmental stages indicated. Note the changes in Nrp1 expression levels and in its expression pattern during development. ( e ) <t>qRT-PCR</t> analysis of Nrp1 in microdissected MN columns at E10.5, E11.5 and E12.5. Data are represented as mean±s.e.m. n =4 individual experiments done in triplicates. ( f – k ) Representative images of ISH for Nrp1 in chicken SC sections at the developmental stages indicated. ( l ) qRT-PCR analysis showing downregulation of Nrp1 , Flt1 ( mFlt1+sFlt1 ), sFlt1 and mFlt1 levels in MN columns of chicken embryos electroporated with Hb9-EGFP-miNRP1#1. Vegf <t>mRNA</t> levels are unaffected. n =7, * P =0.0437 ( Nrp1 ), * P =0.0306 ( Flt1 ), ** P =0.0081 ( sFlt1 ), ** P =0.0055 ( mFlt1 ) and P =ns ( Vegf ). ( m ) Representative image of Hb9-EGFP-miNRP1#1 electroporated chicken embryos showing blood vessel ingression into MN columns in the electroporated side of the SC (yellow arrowhead) but not in the non-electroporated one. ( n ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side in Hb9-EGFP-miNRP1#1 embryos. n =8, * P =0.0274. ( o ) Representative image of rescue experiment, in which Hb9-EGFP-miNRP1#1 is co-electroporated with a mouse sFlt1-HA plasmid. ( p ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side. n =6, * P =0.0349. Scale bars 100 μm.
    Mrna Expression Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of miR-200b-3p, miR-200c-3p, and their downstream target ZEB2 mRNA in carcinoma tissues of non-IBC and IBC in comparison with normal breast tissue samples. ( A ) miR-200b-3p and ( B ) miR-200c-3p expression levels are significantly repressed in carcinoma tissues of IBC relative to non-IBC as determined by qPCR, whereas their expression levels in non-IBC and IBC are elevated when compared to normal breast tissue samples. miR-200b-3p and miR-200c-3p expression are log2-transformed and normalized to values of normal tissues collected during reduction mammoplasty. # p

    Journal: Biomolecules

    Article Title: Inflammatory Breast Carcinoma: Elevated microRNA miR-181b-5p and Reduced miR-200b-3p, miR-200c-3p, and miR-203a-3p Expression as Potential Biomarkers with Diagnostic Value

    doi: 10.3390/biom10071059

    Figure Lengend Snippet: Expression of miR-200b-3p, miR-200c-3p, and their downstream target ZEB2 mRNA in carcinoma tissues of non-IBC and IBC in comparison with normal breast tissue samples. ( A ) miR-200b-3p and ( B ) miR-200c-3p expression levels are significantly repressed in carcinoma tissues of IBC relative to non-IBC as determined by qPCR, whereas their expression levels in non-IBC and IBC are elevated when compared to normal breast tissue samples. miR-200b-3p and miR-200c-3p expression are log2-transformed and normalized to values of normal tissues collected during reduction mammoplasty. # p

    Article Snippet: For ZEB2 mRNA expression-level detection, total RNA isolated from primary normal breast tissues or breast carcinoma tissues was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo scientific, ON, Canada) and qPCR of ZEB2 mRNA expression was performed using Brilliant SYBR Green qPCR master mix (Applied Biosystems, CA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay

    Functionality tests of the GFP-tagged fTRG lines by genetic complementation analysis. ( A ) Genetic rescue statistics of null/strong mutant alleles for 46 selected fTRG lines. Note that more than two-thirds of the lines show a rescue (see Table 2 ). ( B , C ) osk -GFP mRNA (in yellow) expressed from fTRG1394 rescues egg-chamber development of an osk null allele ( Jenny et al., 2006 ). osk -GFP mRNA enriches in the early oocyte ( B , stage 6) and rescues the oogenesis arrest and the DNA condensation defect of the osk mutant ( B’ , yellow arrowhead). At stage 10 osk -GFP RNA enriches at the posterior pole ( C ) and produces sufficient protein to ensure proper embryogenesis. osk -GFP mRNA is shown in yellow, DAPI in magenta; scale bars indicate 30 µm. DOI: http://dx.doi.org/10.7554/eLife.12068.006

    Journal: eLife

    Article Title: A genome-wide resource for the analysis of protein localisation in Drosophila

    doi: 10.7554/eLife.12068

    Figure Lengend Snippet: Functionality tests of the GFP-tagged fTRG lines by genetic complementation analysis. ( A ) Genetic rescue statistics of null/strong mutant alleles for 46 selected fTRG lines. Note that more than two-thirds of the lines show a rescue (see Table 2 ). ( B , C ) osk -GFP mRNA (in yellow) expressed from fTRG1394 rescues egg-chamber development of an osk null allele ( Jenny et al., 2006 ). osk -GFP mRNA enriches in the early oocyte ( B , stage 6) and rescues the oogenesis arrest and the DNA condensation defect of the osk mutant ( B’ , yellow arrowhead). At stage 10 osk -GFP RNA enriches at the posterior pole ( C ) and produces sufficient protein to ensure proper embryogenesis. osk -GFP mRNA is shown in yellow, DAPI in magenta; scale bars indicate 30 µm. DOI: http://dx.doi.org/10.7554/eLife.12068.006

    Article Snippet: Detection of the oskar-GFP mRNA was performed with a gfp -antisense probe ( ) and co-staining of osk mRNA and Osk protein was done as previously described ( ) using a gfp -antisense probe and a rabbit anti-GFP antibody (1:1000, ThermoFisher).

    Techniques: Mutagenesis

    Posttranscriptional regulation of protein expression during oogenesis. ( A , B ) osk-GFP mRNA visualised by an anti-GFP labelled RNA probe (yellow, DAPI in magenta) at stage 6 and stage 10 of oogenesis. ( A’ , B’ ) Osk-GFP protein visualised by anti-GFP antibody (green, DAPI in magenta) at stage 6 and stage 10. Note that Osk-GFP protein is not detectable at stage 6. ( C , D ) corolla-GFP mRNA (yellow, DAPI in magenta) at stage 6 and stage 8. ( E , F ) Corolla-GFP protein (green, DAPI in magenta) at stage 6 and stage 8. Note that Corolla-GFP protein is only detectable at stage 6 but not stage 8. Scale bars indicate 30 µm. DOI: http://dx.doi.org/10.7554/eLife.12068.010

    Journal: eLife

    Article Title: A genome-wide resource for the analysis of protein localisation in Drosophila

    doi: 10.7554/eLife.12068

    Figure Lengend Snippet: Posttranscriptional regulation of protein expression during oogenesis. ( A , B ) osk-GFP mRNA visualised by an anti-GFP labelled RNA probe (yellow, DAPI in magenta) at stage 6 and stage 10 of oogenesis. ( A’ , B’ ) Osk-GFP protein visualised by anti-GFP antibody (green, DAPI in magenta) at stage 6 and stage 10. Note that Osk-GFP protein is not detectable at stage 6. ( C , D ) corolla-GFP mRNA (yellow, DAPI in magenta) at stage 6 and stage 8. ( E , F ) Corolla-GFP protein (green, DAPI in magenta) at stage 6 and stage 8. Note that Corolla-GFP protein is only detectable at stage 6 but not stage 8. Scale bars indicate 30 µm. DOI: http://dx.doi.org/10.7554/eLife.12068.010

    Article Snippet: Detection of the oskar-GFP mRNA was performed with a gfp -antisense probe ( ) and co-staining of osk mRNA and Osk protein was done as previously described ( ) using a gfp -antisense probe and a rabbit anti-GFP antibody (1:1000, ThermoFisher).

    Techniques: Expressing

    RIOK2 and NOB1 mRNA expression levels in NSCLC tissues compared with normal tissues. ( a ) The mean RIOK2 mRNA expression level was higher in the tumour tissues (2.16 ± 0.57) than in the corresponding paired adjacent normal lung tissues (0.46 ± 0.21) ( P

    Journal: Scientific Reports

    Article Title: High Expression of RIOK2 and NOB1 Predict Human Non-small Cell Lung Cancer Outcomes

    doi: 10.1038/srep28666

    Figure Lengend Snippet: RIOK2 and NOB1 mRNA expression levels in NSCLC tissues compared with normal tissues. ( a ) The mean RIOK2 mRNA expression level was higher in the tumour tissues (2.16 ± 0.57) than in the corresponding paired adjacent normal lung tissues (0.46 ± 0.21) ( P

    Article Snippet: The RIOK2 and NOB1 mRNA levels were detected with a one-step RT-qPCR reaction using a SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.

    Techniques: Expressing

    RIOK2 and NOB1 mRNA expression levels in NSCLC cells. ( a ) The RIOK2 mRNA level in the NSCLC cell lines (A549, H1299, H1975 and H1650) was higher than that in the normal lung cell line (BEAS-2B); ( b ) The NOB1 mRNA level in the NSCLC cell lines (A549, H1299, H1975 and H1650) was higher than that in the normal lung cell line (BEAS-2B).

    Journal: Scientific Reports

    Article Title: High Expression of RIOK2 and NOB1 Predict Human Non-small Cell Lung Cancer Outcomes

    doi: 10.1038/srep28666

    Figure Lengend Snippet: RIOK2 and NOB1 mRNA expression levels in NSCLC cells. ( a ) The RIOK2 mRNA level in the NSCLC cell lines (A549, H1299, H1975 and H1650) was higher than that in the normal lung cell line (BEAS-2B); ( b ) The NOB1 mRNA level in the NSCLC cell lines (A549, H1299, H1975 and H1650) was higher than that in the normal lung cell line (BEAS-2B).

    Article Snippet: The RIOK2 and NOB1 mRNA levels were detected with a one-step RT-qPCR reaction using a SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.

    Techniques: Expressing

    NRP1 regulates sFlt1 expression and as a consequence blood vessel guidance. ( a – d ) Representative images of ISH for Nrp1 in mouse SC sections at the developmental stages indicated. Note the changes in Nrp1 expression levels and in its expression pattern during development. ( e ) qRT-PCR analysis of Nrp1 in microdissected MN columns at E10.5, E11.5 and E12.5. Data are represented as mean±s.e.m. n =4 individual experiments done in triplicates. ( f – k ) Representative images of ISH for Nrp1 in chicken SC sections at the developmental stages indicated. ( l ) qRT-PCR analysis showing downregulation of Nrp1 , Flt1 ( mFlt1+sFlt1 ), sFlt1 and mFlt1 levels in MN columns of chicken embryos electroporated with Hb9-EGFP-miNRP1#1. Vegf mRNA levels are unaffected. n =7, * P =0.0437 ( Nrp1 ), * P =0.0306 ( Flt1 ), ** P =0.0081 ( sFlt1 ), ** P =0.0055 ( mFlt1 ) and P =ns ( Vegf ). ( m ) Representative image of Hb9-EGFP-miNRP1#1 electroporated chicken embryos showing blood vessel ingression into MN columns in the electroporated side of the SC (yellow arrowhead) but not in the non-electroporated one. ( n ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side in Hb9-EGFP-miNRP1#1 embryos. n =8, * P =0.0274. ( o ) Representative image of rescue experiment, in which Hb9-EGFP-miNRP1#1 is co-electroporated with a mouse sFlt1-HA plasmid. ( p ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side. n =6, * P =0.0349. Scale bars 100 μm.

    Journal: Nature Communications

    Article Title: Motor neurons control blood vessel patterning in the developing spinal cord

    doi: 10.1038/ncomms14583

    Figure Lengend Snippet: NRP1 regulates sFlt1 expression and as a consequence blood vessel guidance. ( a – d ) Representative images of ISH for Nrp1 in mouse SC sections at the developmental stages indicated. Note the changes in Nrp1 expression levels and in its expression pattern during development. ( e ) qRT-PCR analysis of Nrp1 in microdissected MN columns at E10.5, E11.5 and E12.5. Data are represented as mean±s.e.m. n =4 individual experiments done in triplicates. ( f – k ) Representative images of ISH for Nrp1 in chicken SC sections at the developmental stages indicated. ( l ) qRT-PCR analysis showing downregulation of Nrp1 , Flt1 ( mFlt1+sFlt1 ), sFlt1 and mFlt1 levels in MN columns of chicken embryos electroporated with Hb9-EGFP-miNRP1#1. Vegf mRNA levels are unaffected. n =7, * P =0.0437 ( Nrp1 ), * P =0.0306 ( Flt1 ), ** P =0.0081 ( sFlt1 ), ** P =0.0055 ( mFlt1 ) and P =ns ( Vegf ). ( m ) Representative image of Hb9-EGFP-miNRP1#1 electroporated chicken embryos showing blood vessel ingression into MN columns in the electroporated side of the SC (yellow arrowhead) but not in the non-electroporated one. ( n ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side in Hb9-EGFP-miNRP1#1 embryos. n =8, * P =0.0274. ( o ) Representative image of rescue experiment, in which Hb9-EGFP-miNRP1#1 is co-electroporated with a mouse sFlt1-HA plasmid. ( p ) Quantification of blood vessel density in MN columns (Isl1/2 + ) shown as ratio between electroporated and non-electroporated side. n =6, * P =0.0349. Scale bars 100 μm.

    Article Snippet: Subsequently, DNAseI-treated (EN0521, Thermo Scientific) RNA was reverse-transcribed into cDNA using either Maxima Reverse Transcriptase (EP0742, Thermo Scientific) or SuperScriptVilo (11754-050, Thermo Scientific). mRNA expression levels were assessed by qRT-PCR using Fast SYBR Green Master Mix (00408995, Thermo Scientific), relative to the expression level of Gapdh or 18S (for mouse and chicken samples, respectively).

    Techniques: Expressing, In Situ Hybridization, Quantitative RT-PCR, Plasmid Preparation