sense antisense primer pairs  (Qiagen)

 
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    Name:
    QIAGEN LongRange PCR Kit
    Description:
    For sensitive and accurate long range PCR Kit contents Qiagen LongRange PCR Kit 20 x 50L rxns Amplification of Extremely Long PCR Products up to 40 kb DNA Low Error Rates Ensured by High fidelity Enzyme Minimal PCR Optimization due to Unique Buffer System Amplification of Low copy Targets and GC rich Templates For Sensitive and Accurate Long range PCR Includes LongRange PCR Enzyme Mix 40U LongRange PCR Buffer 5x Q Solution RNase free Water 10mM dNTPs Benefits Amplification of extremely long PCR products up to 40 kb DNA Low error rates ensured by high fidelity enzyme Minimal PCR optimization due to unique buffer system Amplification of low copy targets and GC rich templates
    Catalog Number:
    206401
    Price:
    66.9
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    QIAGEN LongRange PCR Kit
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    Structured Review

    Qiagen sense antisense primer pairs
    QIAGEN LongRange PCR Kit
    For sensitive and accurate long range PCR Kit contents Qiagen LongRange PCR Kit 20 x 50L rxns Amplification of Extremely Long PCR Products up to 40 kb DNA Low Error Rates Ensured by High fidelity Enzyme Minimal PCR Optimization due to Unique Buffer System Amplification of Low copy Targets and GC rich Templates For Sensitive and Accurate Long range PCR Includes LongRange PCR Enzyme Mix 40U LongRange PCR Buffer 5x Q Solution RNase free Water 10mM dNTPs Benefits Amplification of extremely long PCR products up to 40 kb DNA Low error rates ensured by high fidelity enzyme Minimal PCR optimization due to unique buffer system Amplification of low copy targets and GC rich templates
    https://www.bioz.com/result/sense antisense primer pairs/product/Qiagen
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    Images

    1) Product Images from "New Fusion Transcripts Identified in Normal Karyotype Acute Myeloid Leukemia"

    Article Title: New Fusion Transcripts Identified in Normal Karyotype Acute Myeloid Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051203

    Validation of sense and antisense fusion transcripts by strand-specific RT-PCR. A. Summary for RNA samples from 8 myeloid cell lines. B. CIITA-DEXI sense and antisense fusion transcripts detected in myeloid cell lines and AML samples. +: positive control with beta-actin; -: netative control without RNA templates.
    Figure Legend Snippet: Validation of sense and antisense fusion transcripts by strand-specific RT-PCR. A. Summary for RNA samples from 8 myeloid cell lines. B. CIITA-DEXI sense and antisense fusion transcripts detected in myeloid cell lines and AML samples. +: positive control with beta-actin; -: netative control without RNA templates.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Positive Control

    2) Product Images from "ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones"

    Article Title: ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031977

    Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.
    Figure Legend Snippet: Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Transfection, Expressing, Polymerase Chain Reaction, Amplification, Positive Control

    3) Product Images from "Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection"

    Article Title: Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181383

    RT-PCR analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).
    Figure Legend Snippet: RT-PCR analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    4) Product Images from "The neuronal K+Cl− co-transporter 2 (Slc12a5) modulates insulin secretion"

    Article Title: The neuronal K+Cl− co-transporter 2 (Slc12a5) modulates insulin secretion

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01814-0

    KCC2 is expressed in rodent β-cell lines and pancreatic islets. ( A – D ) Original ethidium bromide stained gels (inverted) showing KCC2 mRNA expression in the mouse β-cell line MIN6 ( A ), human ( B ) and mouse islets ( C ) and the rat INS1-E β-cell line ( D ). Shown are PCR products of expected sizes representing KCC2a and KCC2b. As the positive control of RT-PCRs, transcripts of GAPDH were amplified (555 bp). As the negative control, water was used instead of total cDNA. ( E ) Partial chromatograms obtained from representative DNA sequencing reactions using purified KCC2a-671 and KCC2b-602 PCR products from MIN6 ( black asterisks ). The DNA sequences obtained are 100% identical to the spliced version of KCC2a and KCC2b, respectively. ( F ) Representative original 1% agarose gel digital image cropped to include bands of relevant sizes. RT coupled to long-range PCR using total RNA from MIN6 and primer sets KCC2a-3591 or KCC2b-4024 (Supplementary Table 1 ). The PCR products of 3591 bp (KCC2a) and 4024 bp (KCC2b) were purified from this gel, directly cloned in pCR-Blunt II-TOPO vectors and fully sequenced in both directions. ( G ) MIN6 and mouse brain KCC2a and KCC2b mRNA quantification ( n = 5). Results are expressed relative to MIN6 KCC2a. ( H ) Cropped digitised immunoblots of the indicated protein extracts (µg) obtained from MIN6, mouse (m) and human (h) islets and mouse brain. ( I ) Digitized immunoblot cropped to show KCC2 protein expression in purified plasma membranes of COS7 cells and MIN6 β-cells. ( J,K ) Confocal images of MIN6 β-cells immunolabeled using validated KCC2 antibodies. The square in ( J ) is shown at higher magnification in K to indicate with white arrows immunoreactive KCC2 towards the edges of the cells. Nuclei have been counterstained with DAPI. Scale bar represents 10 µm.
    Figure Legend Snippet: KCC2 is expressed in rodent β-cell lines and pancreatic islets. ( A – D ) Original ethidium bromide stained gels (inverted) showing KCC2 mRNA expression in the mouse β-cell line MIN6 ( A ), human ( B ) and mouse islets ( C ) and the rat INS1-E β-cell line ( D ). Shown are PCR products of expected sizes representing KCC2a and KCC2b. As the positive control of RT-PCRs, transcripts of GAPDH were amplified (555 bp). As the negative control, water was used instead of total cDNA. ( E ) Partial chromatograms obtained from representative DNA sequencing reactions using purified KCC2a-671 and KCC2b-602 PCR products from MIN6 ( black asterisks ). The DNA sequences obtained are 100% identical to the spliced version of KCC2a and KCC2b, respectively. ( F ) Representative original 1% agarose gel digital image cropped to include bands of relevant sizes. RT coupled to long-range PCR using total RNA from MIN6 and primer sets KCC2a-3591 or KCC2b-4024 (Supplementary Table 1 ). The PCR products of 3591 bp (KCC2a) and 4024 bp (KCC2b) were purified from this gel, directly cloned in pCR-Blunt II-TOPO vectors and fully sequenced in both directions. ( G ) MIN6 and mouse brain KCC2a and KCC2b mRNA quantification ( n = 5). Results are expressed relative to MIN6 KCC2a. ( H ) Cropped digitised immunoblots of the indicated protein extracts (µg) obtained from MIN6, mouse (m) and human (h) islets and mouse brain. ( I ) Digitized immunoblot cropped to show KCC2 protein expression in purified plasma membranes of COS7 cells and MIN6 β-cells. ( J,K ) Confocal images of MIN6 β-cells immunolabeled using validated KCC2 antibodies. The square in ( J ) is shown at higher magnification in K to indicate with white arrows immunoreactive KCC2 towards the edges of the cells. Nuclei have been counterstained with DAPI. Scale bar represents 10 µm.

    Techniques Used: Staining, Expressing, Polymerase Chain Reaction, Positive Control, Amplification, Negative Control, DNA Sequencing, Purification, Agarose Gel Electrophoresis, Clone Assay, Western Blot, Immunolabeling

    KCC2-S25 is expressed in MIN6 β-cells, human islets and mouse pancreas. ( A ) Representation of KCC2a/b amplicons obtained by using the KCC2-565 primer set. Indicated are the MspI restriction sites and the predicted length of the digestion products in bp. Exon 25 is highlighted in red. Its splicing eliminates an MspI site in the amplicon. ( B ) Ethidium bormide stained gel, inverted from its original gray-scale digital picture, showing RT-PCR products of expected size (565 bp) obtained by using the primer set KCC2-565 and total RNA from mouse spinal cord, brain and MIN6 β-cells. As negative control, water was used instead of total cDNA. ( C ) Representative ethidium bromide stained 2% agarose gel inverted from original where MspI -digested PCR products were separated. Also shown, representative densitometry analysis of the MspI banding pattern to estimate the relative contribution of KCC2-S25 (~54%) to the total KCC2 pool. ( D ) Representative ethidium bromide stained gel inverted from original showing an RT-PCR experiment performed using mouse islet RNA and the KCC2-565 primer set. Note the product of expected size and MspI digestion analysis of restriction fragments. ( E ) Representative ethidium bormide stained gel inverted from original showing RT-PCR experiment using total RNA from human islets and the KCC2-657 primer to obtain amplicons of expected size (657 bp) and a posteriori MspI digestion analysis. ( F ) Expression levels of total KCC2 in adult mouse brain using qPCR primers that do not distinguish among known KCC2 variants (total KCC2) or specific to exon 25 (KCC2a/b). ( G ) Representation of human KCC2a/b amplicons obtained using KCC2-657 primer set and predicted MspI restriction fragments for KCC2a/b-S25 (176 bp) and KCC2a/b (102 bp + 89 bp).
    Figure Legend Snippet: KCC2-S25 is expressed in MIN6 β-cells, human islets and mouse pancreas. ( A ) Representation of KCC2a/b amplicons obtained by using the KCC2-565 primer set. Indicated are the MspI restriction sites and the predicted length of the digestion products in bp. Exon 25 is highlighted in red. Its splicing eliminates an MspI site in the amplicon. ( B ) Ethidium bormide stained gel, inverted from its original gray-scale digital picture, showing RT-PCR products of expected size (565 bp) obtained by using the primer set KCC2-565 and total RNA from mouse spinal cord, brain and MIN6 β-cells. As negative control, water was used instead of total cDNA. ( C ) Representative ethidium bromide stained 2% agarose gel inverted from original where MspI -digested PCR products were separated. Also shown, representative densitometry analysis of the MspI banding pattern to estimate the relative contribution of KCC2-S25 (~54%) to the total KCC2 pool. ( D ) Representative ethidium bromide stained gel inverted from original showing an RT-PCR experiment performed using mouse islet RNA and the KCC2-565 primer set. Note the product of expected size and MspI digestion analysis of restriction fragments. ( E ) Representative ethidium bormide stained gel inverted from original showing RT-PCR experiment using total RNA from human islets and the KCC2-657 primer to obtain amplicons of expected size (657 bp) and a posteriori MspI digestion analysis. ( F ) Expression levels of total KCC2 in adult mouse brain using qPCR primers that do not distinguish among known KCC2 variants (total KCC2) or specific to exon 25 (KCC2a/b). ( G ) Representation of human KCC2a/b amplicons obtained using KCC2-657 primer set and predicted MspI restriction fragments for KCC2a/b-S25 (176 bp) and KCC2a/b (102 bp + 89 bp).

    Techniques Used: Amplification, Staining, Reverse Transcription Polymerase Chain Reaction, Negative Control, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    5) Product Images from "Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy"

    Article Title: Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606230

    Effects of CDDP treatment on the DNA damage pathways, and ATM and p53 inhibition Representative PCR analysis of mATR and mChk1 expression in cochleae, testis, and kidney tissues from P3 mice. Water was used as a negative control. Representative Western blot analysis showing the level of Chk1 phosphorylation in protein extracts from cultured whole cochleae treated or not with 10 μM CDDP and from control and UV light‐exposed mouse embryonic fibroblasts (MEF). 3D images showing IHC and OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone (control) or 10 μM CDDP for 1 day and immunolabeled for p‐ATM (green in C) or p‐Chk2 (green in D). Scale bar = 5 μm. 3D images showing OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 1 day and immunolabeled for p‐ATM (green). Scale bar = 5 μm. Quantification of p‐ATM foci number per nucleus in both inner (red bars) and outer (blue bars) hair cells for all treatment conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P ≤ 0.008, *** P ≤ 0.0006; CDDP versus control or CDDP versus CDDP + KU55933). Confocal images showing the basal region of organ of Corti cultures treated with medium alone, 100 μM PFT‐α, 10 μM CDDP, or 10 μM CDDP in combination with 100 μM PFT‐α for 5 days and immunolabeled for myosin 7A (red). Scale bar = 24 μm. Histograms representing the levels of surviving IHCs (red bars) and OHCs (blue bars) for all conditions after 5 days ( n = 5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (* P = 0.02, ** P ≤ 0.006, *** P = 0.0004; CDDP versus control, or CDDP versus CDDP + PFT‐α). Data information: All experiments were performed in triplicate.
    Figure Legend Snippet: Effects of CDDP treatment on the DNA damage pathways, and ATM and p53 inhibition Representative PCR analysis of mATR and mChk1 expression in cochleae, testis, and kidney tissues from P3 mice. Water was used as a negative control. Representative Western blot analysis showing the level of Chk1 phosphorylation in protein extracts from cultured whole cochleae treated or not with 10 μM CDDP and from control and UV light‐exposed mouse embryonic fibroblasts (MEF). 3D images showing IHC and OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone (control) or 10 μM CDDP for 1 day and immunolabeled for p‐ATM (green in C) or p‐Chk2 (green in D). Scale bar = 5 μm. 3D images showing OHC nuclei from the basal region of the organ of Corti cultures treated with medium alone, 10 μM KU55933, 10 μM CDDP, or 10 μM CDDP in combination with 10 μM KU55933 for 1 day and immunolabeled for p‐ATM (green). Scale bar = 5 μm. Quantification of p‐ATM foci number per nucleus in both inner (red bars) and outer (blue bars) hair cells for all treatment conditions ( n = 50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (** P ≤ 0.008, *** P ≤ 0.0006; CDDP versus control or CDDP versus CDDP + KU55933). Confocal images showing the basal region of organ of Corti cultures treated with medium alone, 100 μM PFT‐α, 10 μM CDDP, or 10 μM CDDP in combination with 100 μM PFT‐α for 5 days and immunolabeled for myosin 7A (red). Scale bar = 24 μm. Histograms representing the levels of surviving IHCs (red bars) and OHCs (blue bars) for all conditions after 5 days ( n = 5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One‐way ANOVA test followed by post hoc Tukey's test (* P = 0.02, ** P ≤ 0.006, *** P = 0.0004; CDDP versus control, or CDDP versus CDDP + PFT‐α). Data information: All experiments were performed in triplicate.

    Techniques Used: Inhibition, Polymerase Chain Reaction, Expressing, Mouse Assay, Negative Control, Western Blot, Cell Culture, Immunohistochemistry, Immunolabeling

    6) Product Images from "Insights into the Function and Evolution of Taste 1 Receptor Gene Family in the Carnivore Fish Gilthead Seabream (Sparus aurata)"

    Article Title: Insights into the Function and Evolution of Taste 1 Receptor Gene Family in the Carnivore Fish Gilthead Seabream (Sparus aurata)

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21207732

    Comparisons of proline Log-dose response curves of activation in heterologous transfections of hetero- and homo-dimeric saT1R combinations of saT1R1 / R3 (black dots), saT1R2a / R3 (white dots), saT1R2b / 3 (black triangles), saT1R1 / R1 (white triangles) and saT1R3 / R3 (black squares). Response is measured as RLU mean ± SEM of four independent determinations normalized to the mean response of the same transfections stimulated with reduced assay medium (BME + 1% FBS), expressed as percentage relative to basal levels (BL).
    Figure Legend Snippet: Comparisons of proline Log-dose response curves of activation in heterologous transfections of hetero- and homo-dimeric saT1R combinations of saT1R1 / R3 (black dots), saT1R2a / R3 (white dots), saT1R2b / 3 (black triangles), saT1R1 / R1 (white triangles) and saT1R3 / R3 (black squares). Response is measured as RLU mean ± SEM of four independent determinations normalized to the mean response of the same transfections stimulated with reduced assay medium (BME + 1% FBS), expressed as percentage relative to basal levels (BL).

    Techniques Used: Activation Assay, Transfection

    7) Product Images from "Do anesthetics and sampling strategies affect transcription analysis of fish tissues?"

    Article Title: Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-48

    Transcriptional levels (mean normalized expression MNE) of Na + - K + - ATPase α1b in A) gill and B) brain and of Hypoxia Inducible Factor 1 ( HIF1 ) in C) gill and D) brain tissues of Atlantic salmon differentially treated during sampling. n = 6 in all groups except n = 5 for HIF1 120 min. metacaine, HIF1 120 min. isoeugenol and Na + - K + - ATPase α1b 120 min. groups. For the HIF1 120 min. isoeugenol group, n = 4. Group identity: 0 min. seawater control (0 SW), 30 min. seawater (30 SW), 30 min. isoeugenol (30 I), 120 min. seawater (120 SW), 120 min. metacaine (120 M) and 120 min. isoeugenol (120 I). An * denotes significant differences (P
    Figure Legend Snippet: Transcriptional levels (mean normalized expression MNE) of Na + - K + - ATPase α1b in A) gill and B) brain and of Hypoxia Inducible Factor 1 ( HIF1 ) in C) gill and D) brain tissues of Atlantic salmon differentially treated during sampling. n = 6 in all groups except n = 5 for HIF1 120 min. metacaine, HIF1 120 min. isoeugenol and Na + - K + - ATPase α1b 120 min. groups. For the HIF1 120 min. isoeugenol group, n = 4. Group identity: 0 min. seawater control (0 SW), 30 min. seawater (30 SW), 30 min. isoeugenol (30 I), 120 min. seawater (120 SW), 120 min. metacaine (120 M) and 120 min. isoeugenol (120 I). An * denotes significant differences (P

    Techniques Used: Expressing, Sampling

    8) Product Images from "The neuronal K+Cl− co-transporter 2 (Slc12a5) modulates insulin secretion"

    Article Title: The neuronal K+Cl− co-transporter 2 (Slc12a5) modulates insulin secretion

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01814-0

    KCC2 is expressed in rodent β-cell lines and pancreatic islets. ( A – D ) Original ethidium bromide stained gels (inverted) showing KCC2 mRNA expression in the mouse β-cell line MIN6 ( A ), human ( B ) and mouse islets ( C ) and the rat INS1-E β-cell line ( D ). Shown are PCR products of expected sizes representing KCC2a and KCC2b. As the positive control of RT-PCRs, transcripts of GAPDH were amplified (555 bp). As the negative control, water was used instead of total cDNA. ( E ) Partial chromatograms obtained from representative DNA sequencing reactions using purified KCC2a-671 and KCC2b-602 PCR products from MIN6 ( black asterisks ). The DNA sequences obtained are 100% identical to the spliced version of KCC2a and KCC2b, respectively. ( F ) Representative original 1% agarose gel digital image cropped to include bands of relevant sizes. RT coupled to long-range PCR using total RNA from MIN6 and primer sets KCC2a-3591 or KCC2b-4024 (Supplementary Table 1 ). The PCR products of 3591 bp (KCC2a) and 4024 bp (KCC2b) were purified from this gel, directly cloned in pCR-Blunt II-TOPO vectors and fully sequenced in both directions. ( G ) MIN6 and mouse brain KCC2a and KCC2b mRNA quantification ( n = 5). Results are expressed relative to MIN6 KCC2a. ( H ) Cropped digitised immunoblots of the indicated protein extracts (µg) obtained from MIN6, mouse (m) and human (h) islets and mouse brain. ( I ) Digitized immunoblot cropped to show KCC2 protein expression in purified plasma membranes of COS7 cells and MIN6 β-cells. ( J,K ) Confocal images of MIN6 β-cells immunolabeled using validated KCC2 antibodies. The square in ( J ) is shown at higher magnification in K to indicate with white arrows immunoreactive KCC2 towards the edges of the cells. Nuclei have been counterstained with DAPI. Scale bar represents 10 µm.
    Figure Legend Snippet: KCC2 is expressed in rodent β-cell lines and pancreatic islets. ( A – D ) Original ethidium bromide stained gels (inverted) showing KCC2 mRNA expression in the mouse β-cell line MIN6 ( A ), human ( B ) and mouse islets ( C ) and the rat INS1-E β-cell line ( D ). Shown are PCR products of expected sizes representing KCC2a and KCC2b. As the positive control of RT-PCRs, transcripts of GAPDH were amplified (555 bp). As the negative control, water was used instead of total cDNA. ( E ) Partial chromatograms obtained from representative DNA sequencing reactions using purified KCC2a-671 and KCC2b-602 PCR products from MIN6 ( black asterisks ). The DNA sequences obtained are 100% identical to the spliced version of KCC2a and KCC2b, respectively. ( F ) Representative original 1% agarose gel digital image cropped to include bands of relevant sizes. RT coupled to long-range PCR using total RNA from MIN6 and primer sets KCC2a-3591 or KCC2b-4024 (Supplementary Table 1 ). The PCR products of 3591 bp (KCC2a) and 4024 bp (KCC2b) were purified from this gel, directly cloned in pCR-Blunt II-TOPO vectors and fully sequenced in both directions. ( G ) MIN6 and mouse brain KCC2a and KCC2b mRNA quantification ( n = 5). Results are expressed relative to MIN6 KCC2a. ( H ) Cropped digitised immunoblots of the indicated protein extracts (µg) obtained from MIN6, mouse (m) and human (h) islets and mouse brain. ( I ) Digitized immunoblot cropped to show KCC2 protein expression in purified plasma membranes of COS7 cells and MIN6 β-cells. ( J,K ) Confocal images of MIN6 β-cells immunolabeled using validated KCC2 antibodies. The square in ( J ) is shown at higher magnification in K to indicate with white arrows immunoreactive KCC2 towards the edges of the cells. Nuclei have been counterstained with DAPI. Scale bar represents 10 µm.

    Techniques Used: Staining, Expressing, Polymerase Chain Reaction, Positive Control, Amplification, Negative Control, DNA Sequencing, Purification, Agarose Gel Electrophoresis, Clone Assay, Western Blot, Immunolabeling

    9) Product Images from "New Fusion Transcripts Identified in Normal Karyotype Acute Myeloid Leukemia"

    Article Title: New Fusion Transcripts Identified in Normal Karyotype Acute Myeloid Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051203

    Validation of sense and antisense fusion transcripts by strand-specific RT-PCR. A. Summary for RNA samples from 8 myeloid cell lines. B. CIITA-DEXI sense and antisense fusion transcripts detected in myeloid cell lines and AML samples. +: positive control with beta-actin; -: netative control without RNA templates.
    Figure Legend Snippet: Validation of sense and antisense fusion transcripts by strand-specific RT-PCR. A. Summary for RNA samples from 8 myeloid cell lines. B. CIITA-DEXI sense and antisense fusion transcripts detected in myeloid cell lines and AML samples. +: positive control with beta-actin; -: netative control without RNA templates.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Positive Control

    10) Product Images from "Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus"

    Article Title: Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121656

    Expression analysis of GUS transcripts under the control of Cotton leaf curl Burewala virus promoters in transformed Nicotiana tabacum plants. (A) RT-PCR amplification of GUS transcripts from total RNA isolated from N . tabacum plants transformed by CLCuBuV Rep (1 and 2), CLCuBuV CP (3 and 4) and CaMV 35S (5 and 6) promoter constructs, wild type control tobacco plant (7). Electrophoresis of RT-PCR amplification of actin transcripts isolated from total RNA from transformed N . tabacum plants generated from the same constructs. (B) The relative ratio of GUS transcripts quantified by Reverse transcription quantitative real-time PCR. All the experiments were repeated three times with several replicates. Error bars indicate SE.
    Figure Legend Snippet: Expression analysis of GUS transcripts under the control of Cotton leaf curl Burewala virus promoters in transformed Nicotiana tabacum plants. (A) RT-PCR amplification of GUS transcripts from total RNA isolated from N . tabacum plants transformed by CLCuBuV Rep (1 and 2), CLCuBuV CP (3 and 4) and CaMV 35S (5 and 6) promoter constructs, wild type control tobacco plant (7). Electrophoresis of RT-PCR amplification of actin transcripts isolated from total RNA from transformed N . tabacum plants generated from the same constructs. (B) The relative ratio of GUS transcripts quantified by Reverse transcription quantitative real-time PCR. All the experiments were repeated three times with several replicates. Error bars indicate SE.

    Techniques Used: Expressing, Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation, Construct, Electrophoresis, Generated, Real-time Polymerase Chain Reaction

    11) Product Images from "ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones"

    Article Title: ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031977

    Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.
    Figure Legend Snippet: Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Transfection, Expressing, Polymerase Chain Reaction, Amplification, Positive Control

    12) Product Images from "The neuronal K+Cl− co-transporter 2 (Slc12a5) modulates insulin secretion"

    Article Title: The neuronal K+Cl− co-transporter 2 (Slc12a5) modulates insulin secretion

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01814-0

    KCC2 is expressed in rodent β-cell lines and pancreatic islets. ( A – D ) Original ethidium bromide stained gels (inverted) showing KCC2 mRNA expression in the mouse β-cell line MIN6 ( A ), human ( B ) and mouse islets ( C ) and the rat INS1-E β-cell line ( D ). Shown are PCR products of expected sizes representing KCC2a and KCC2b. As the positive control of RT-PCRs, transcripts of GAPDH were amplified (555 bp). As the negative control, water was used instead of total cDNA. ( E ) Partial chromatograms obtained from representative DNA sequencing reactions using purified KCC2a-671 and KCC2b-602 PCR products from MIN6 ( black asterisks ). The DNA sequences obtained are 100% identical to the spliced version of KCC2a and KCC2b, respectively. ( F ) Representative original 1% agarose gel digital image cropped to include bands of relevant sizes. RT coupled to long-range PCR using total RNA from MIN6 and primer sets KCC2a-3591 or KCC2b-4024 (Supplementary Table 1 ). The PCR products of 3591 bp (KCC2a) and 4024 bp (KCC2b) were purified from this gel, directly cloned in pCR-Blunt II-TOPO vectors and fully sequenced in both directions. ( G ) MIN6 and mouse brain KCC2a and KCC2b mRNA quantification ( n = 5). Results are expressed relative to MIN6 KCC2a. ( H ) Cropped digitised immunoblots of the indicated protein extracts (µg) obtained from MIN6, mouse (m) and human (h) islets and mouse brain. ( I ) Digitized immunoblot cropped to show KCC2 protein expression in purified plasma membranes of COS7 cells and MIN6 β-cells. ( J,K ) Confocal images of MIN6 β-cells immunolabeled using validated KCC2 antibodies. The square in ( J ) is shown at higher magnification in K to indicate with white arrows immunoreactive KCC2 towards the edges of the cells. Nuclei have been counterstained with DAPI. Scale bar represents 10 µm.
    Figure Legend Snippet: KCC2 is expressed in rodent β-cell lines and pancreatic islets. ( A – D ) Original ethidium bromide stained gels (inverted) showing KCC2 mRNA expression in the mouse β-cell line MIN6 ( A ), human ( B ) and mouse islets ( C ) and the rat INS1-E β-cell line ( D ). Shown are PCR products of expected sizes representing KCC2a and KCC2b. As the positive control of RT-PCRs, transcripts of GAPDH were amplified (555 bp). As the negative control, water was used instead of total cDNA. ( E ) Partial chromatograms obtained from representative DNA sequencing reactions using purified KCC2a-671 and KCC2b-602 PCR products from MIN6 ( black asterisks ). The DNA sequences obtained are 100% identical to the spliced version of KCC2a and KCC2b, respectively. ( F ) Representative original 1% agarose gel digital image cropped to include bands of relevant sizes. RT coupled to long-range PCR using total RNA from MIN6 and primer sets KCC2a-3591 or KCC2b-4024 (Supplementary Table 1 ). The PCR products of 3591 bp (KCC2a) and 4024 bp (KCC2b) were purified from this gel, directly cloned in pCR-Blunt II-TOPO vectors and fully sequenced in both directions. ( G ) MIN6 and mouse brain KCC2a and KCC2b mRNA quantification ( n = 5). Results are expressed relative to MIN6 KCC2a. ( H ) Cropped digitised immunoblots of the indicated protein extracts (µg) obtained from MIN6, mouse (m) and human (h) islets and mouse brain. ( I ) Digitized immunoblot cropped to show KCC2 protein expression in purified plasma membranes of COS7 cells and MIN6 β-cells. ( J,K ) Confocal images of MIN6 β-cells immunolabeled using validated KCC2 antibodies. The square in ( J ) is shown at higher magnification in K to indicate with white arrows immunoreactive KCC2 towards the edges of the cells. Nuclei have been counterstained with DAPI. Scale bar represents 10 µm.

    Techniques Used: Staining, Expressing, Polymerase Chain Reaction, Positive Control, Amplification, Negative Control, DNA Sequencing, Purification, Agarose Gel Electrophoresis, Clone Assay, Western Blot, Immunolabeling

    KCC2-S25 is expressed in MIN6 β-cells, human islets and mouse pancreas. ( A ) Representation of KCC2a/b amplicons obtained by using the KCC2-565 primer set. Indicated are the MspI restriction sites and the predicted length of the digestion products in bp. Exon 25 is highlighted in red. Its splicing eliminates an MspI site in the amplicon. ( B ) Ethidium bormide stained gel, inverted from its original gray-scale digital picture, showing RT-PCR products of expected size (565 bp) obtained by using the primer set KCC2-565 and total RNA from mouse spinal cord, brain and MIN6 β-cells. As negative control, water was used instead of total cDNA. ( C ) Representative ethidium bromide stained 2% agarose gel inverted from original where MspI -digested PCR products were separated. Also shown, representative densitometry analysis of the MspI banding pattern to estimate the relative contribution of KCC2-S25 (~54%) to the total KCC2 pool. ( D ) Representative ethidium bromide stained gel inverted from original showing an RT-PCR experiment performed using mouse islet RNA and the KCC2-565 primer set. Note the product of expected size and MspI digestion analysis of restriction fragments. ( E ) Representative ethidium bormide stained gel inverted from original showing RT-PCR experiment using total RNA from human islets and the KCC2-657 primer to obtain amplicons of expected size (657 bp) and a posteriori MspI digestion analysis. ( F ) Expression levels of total KCC2 in adult mouse brain using qPCR primers that do not distinguish among known KCC2 variants (total KCC2) or specific to exon 25 (KCC2a/b). ( G ) Representation of human KCC2a/b amplicons obtained using KCC2-657 primer set and predicted MspI restriction fragments for KCC2a/b-S25 (176 bp) and KCC2a/b (102 bp + 89 bp).
    Figure Legend Snippet: KCC2-S25 is expressed in MIN6 β-cells, human islets and mouse pancreas. ( A ) Representation of KCC2a/b amplicons obtained by using the KCC2-565 primer set. Indicated are the MspI restriction sites and the predicted length of the digestion products in bp. Exon 25 is highlighted in red. Its splicing eliminates an MspI site in the amplicon. ( B ) Ethidium bormide stained gel, inverted from its original gray-scale digital picture, showing RT-PCR products of expected size (565 bp) obtained by using the primer set KCC2-565 and total RNA from mouse spinal cord, brain and MIN6 β-cells. As negative control, water was used instead of total cDNA. ( C ) Representative ethidium bromide stained 2% agarose gel inverted from original where MspI -digested PCR products were separated. Also shown, representative densitometry analysis of the MspI banding pattern to estimate the relative contribution of KCC2-S25 (~54%) to the total KCC2 pool. ( D ) Representative ethidium bromide stained gel inverted from original showing an RT-PCR experiment performed using mouse islet RNA and the KCC2-565 primer set. Note the product of expected size and MspI digestion analysis of restriction fragments. ( E ) Representative ethidium bormide stained gel inverted from original showing RT-PCR experiment using total RNA from human islets and the KCC2-657 primer to obtain amplicons of expected size (657 bp) and a posteriori MspI digestion analysis. ( F ) Expression levels of total KCC2 in adult mouse brain using qPCR primers that do not distinguish among known KCC2 variants (total KCC2) or specific to exon 25 (KCC2a/b). ( G ) Representation of human KCC2a/b amplicons obtained using KCC2-657 primer set and predicted MspI restriction fragments for KCC2a/b-S25 (176 bp) and KCC2a/b (102 bp + 89 bp).

    Techniques Used: Amplification, Staining, Reverse Transcription Polymerase Chain Reaction, Negative Control, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    13) Product Images from "ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones"

    Article Title: ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031977

    Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.
    Figure Legend Snippet: Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Transfection, Expressing, Polymerase Chain Reaction, Amplification, Positive Control

    14) Product Images from "The Replication of Frataxin Gene Is Assured by Activation of Dormant Origins in the Presence of a GAA-Repeat Expansion"

    Article Title: The Replication of Frataxin Gene Is Assured by Activation of Dormant Origins in the Presence of a GAA-Repeat Expansion

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006201

    Two representative images of the FXN locus as detected by FISH and molecular combing. Three probes (green) are used to detect the 850 kb region harboring FXN (red). The position of the GAA-repeat expansion in the mutated alleles is also displayed. Replication tracks are visualized in blue (IdU) and red (CldU), arrows indicate origin positions, the asterisk corresponds to a paused/arrested fork. The two images refer to S8 Fig , molecules 41 and 5 respectively. The region identified by BAC RP11-265B8, where FXN maps, is enlarged to allow a better visualization of origin firing and a sharp interpretation of the replication signals. For each enlargement, the first frame corresponds to the merged image, the second frame shows the probes in green fluorescence, the third one displays the blue and red tracks coinciding with the replicative patterns. Calibration bar = 100 kb.
    Figure Legend Snippet: Two representative images of the FXN locus as detected by FISH and molecular combing. Three probes (green) are used to detect the 850 kb region harboring FXN (red). The position of the GAA-repeat expansion in the mutated alleles is also displayed. Replication tracks are visualized in blue (IdU) and red (CldU), arrows indicate origin positions, the asterisk corresponds to a paused/arrested fork. The two images refer to S8 Fig , molecules 41 and 5 respectively. The region identified by BAC RP11-265B8, where FXN maps, is enlarged to allow a better visualization of origin firing and a sharp interpretation of the replication signals. For each enlargement, the first frame corresponds to the merged image, the second frame shows the probes in green fluorescence, the third one displays the blue and red tracks coinciding with the replicative patterns. Calibration bar = 100 kb.

    Techniques Used: Fluorescence In Situ Hybridization, BAC Assay, Fluorescence

    Replication timing of mutant and normal FXN alleles. Percentages are calculated from pooled data obtained with FRDA (GM15850 and GM16227) and control GM15851 cells. Per each group, at least 550 nuclei were analyzed from at least two independent replicated experiments (Raw data in S1 Table ). SS = nuclei with two single FISH spots (non-replicated alleles); SD = nuclei with one single and one duplicated FISH signal (one allele has been replicated); DD = nuclei with two duplicated FISH signals (both alleles have been replicated); others = nuclei with one or none FISH signals. Error bars indicate standard errors of proportions. The probe used in these experiments is BAC RP11-265B8. For comparison, the replication timing of a late replication sequence ( FRA3B , probe RP11-468L11) in normal GM15851 cells is shown. Examples of FISH replication patterns are shown in the bottom of the Figure.
    Figure Legend Snippet: Replication timing of mutant and normal FXN alleles. Percentages are calculated from pooled data obtained with FRDA (GM15850 and GM16227) and control GM15851 cells. Per each group, at least 550 nuclei were analyzed from at least two independent replicated experiments (Raw data in S1 Table ). SS = nuclei with two single FISH spots (non-replicated alleles); SD = nuclei with one single and one duplicated FISH signal (one allele has been replicated); DD = nuclei with two duplicated FISH signals (both alleles have been replicated); others = nuclei with one or none FISH signals. Error bars indicate standard errors of proportions. The probe used in these experiments is BAC RP11-265B8. For comparison, the replication timing of a late replication sequence ( FRA3B , probe RP11-468L11) in normal GM15851 cells is shown. Examples of FISH replication patterns are shown in the bottom of the Figure.

    Techniques Used: Mutagenesis, Fluorescence In Situ Hybridization, BAC Assay, Sequencing

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    Qiagen long range rt pcr kit
    Characterization of ectopically expressed RNAs by long range <t>RT-PCR.</t> MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα <t>cDNA</t> primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.
    Long Range Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.

    Journal: PLoS ONE

    Article Title: ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

    doi: 10.1371/journal.pone.0031977

    Figure Lengend Snippet: Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.

    Article Snippet: Two µg of total RNA extracted using EZ-RNA isolation kit (Biological Industries, Israel) were transcribed into first strand cDNA by hexamer priming, followed by PCR reactions as specified in the Long range RT-PCR kit (Qiagen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Transfection, Expressing, Polymerase Chain Reaction, Amplification, Positive Control