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Bio-Rad complementary dna cdna
Polymerase chain reaction (PCR) confirmation of gene knockout events in three SCD1 and three THR1 transformants. (A) A 1.3‐kb band representing SCD1 and a 3.7‐kb band representing the transforming <t>DNA</t> were amplified from genomic DNA of the wild‐type (WT) and the three SCD1 transformants, respectively; a 0.6‐kb band representing the SCD1 transcript was amplified from <t>cDNA</t> of the WT only; no band was amplified from RNA samples. (B) A 1.1‐kb band representing THR1 and a 3.8‐kb band representing the transforming DNA were amplified from genomic DNA of the WT and the three THR1 transformants, respectively; a 0.8‐kb band representing the THR1 transcript was amplified from cDNA of the WT only. No band was amplified from RNA samples. (C) A 3.3‐kb band representing the hph gene cassette was amplified from all transformants, but not from the WT. (D) A 0.6‐kb band representing ACT1 was amplified from the WT and all transformants.
Complementary Dna Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth, but not pathogenicity, in Sclerotinia sclerotiorum"

Article Title: Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth, but not pathogenicity, in Sclerotinia sclerotiorum

Journal: Molecular Plant Pathology

doi: 10.1111/mpp.12627

Polymerase chain reaction (PCR) confirmation of gene knockout events in three SCD1 and three THR1 transformants. (A) A 1.3‐kb band representing SCD1 and a 3.7‐kb band representing the transforming DNA were amplified from genomic DNA of the wild‐type (WT) and the three SCD1 transformants, respectively; a 0.6‐kb band representing the SCD1 transcript was amplified from cDNA of the WT only; no band was amplified from RNA samples. (B) A 1.1‐kb band representing THR1 and a 3.8‐kb band representing the transforming DNA were amplified from genomic DNA of the WT and the three THR1 transformants, respectively; a 0.8‐kb band representing the THR1 transcript was amplified from cDNA of the WT only. No band was amplified from RNA samples. (C) A 3.3‐kb band representing the hph gene cassette was amplified from all transformants, but not from the WT. (D) A 0.6‐kb band representing ACT1 was amplified from the WT and all transformants.
Figure Legend Snippet: Polymerase chain reaction (PCR) confirmation of gene knockout events in three SCD1 and three THR1 transformants. (A) A 1.3‐kb band representing SCD1 and a 3.7‐kb band representing the transforming DNA were amplified from genomic DNA of the wild‐type (WT) and the three SCD1 transformants, respectively; a 0.6‐kb band representing the SCD1 transcript was amplified from cDNA of the WT only; no band was amplified from RNA samples. (B) A 1.1‐kb band representing THR1 and a 3.8‐kb band representing the transforming DNA were amplified from genomic DNA of the WT and the three THR1 transformants, respectively; a 0.8‐kb band representing the THR1 transcript was amplified from cDNA of the WT only. No band was amplified from RNA samples. (C) A 3.3‐kb band representing the hph gene cassette was amplified from all transformants, but not from the WT. (D) A 0.6‐kb band representing ACT1 was amplified from the WT and all transformants.

Techniques Used: Polymerase Chain Reaction, Gene Knockout, Amplification

2) Product Images from "Deep Transcriptome Sequencing of Two Green Algae, Chara vulgaris and Chlamydomonas reinhardtii, Provides No Evidence of Organellar RNA Editing"

Article Title: Deep Transcriptome Sequencing of Two Green Algae, Chara vulgaris and Chlamydomonas reinhardtii, Provides No Evidence of Organellar RNA Editing

Journal: Genes

doi: 10.3390/genes8020080

Alignment of the deep sequenced mitochondrial transcriptome of Chlamydomonas reinhardtii strain cc503. ( A ) A single putative RNA editing site was detected in the cytochrome oxidase b gene at nucleotide 1,182. The mean coverage of the cob gene was 28,371.5 ± 7,598 with a min:max of 816:42,177. ( B ) Sequencing of the cob DNA and cDNA from the cc503 isolate used for the deep sequencing revealed a SNP at that site, therefore no editing had taken place.
Figure Legend Snippet: Alignment of the deep sequenced mitochondrial transcriptome of Chlamydomonas reinhardtii strain cc503. ( A ) A single putative RNA editing site was detected in the cytochrome oxidase b gene at nucleotide 1,182. The mean coverage of the cob gene was 28,371.5 ± 7,598 with a min:max of 816:42,177. ( B ) Sequencing of the cob DNA and cDNA from the cc503 isolate used for the deep sequencing revealed a SNP at that site, therefore no editing had taken place.

Techniques Used: Sequencing

Three follow-up experiments attempting to detect three putative RNA editing sites in the C. vulgaris psbI mRNA. ( A ) Chara was grown in nine different environmental conditions: 20 °C, 25 °C, 30 °C and pH 6, 7, 8, in triplicate, and then DNA + RNA were extracted from each. The melt temperatures for genomic DNA (gDNA) and complementary DNA (cDNA) were determined as paired reactions using high resolution melt analysis. Melt temperature differences between gDNA and cDNA derived amplicons were calculated by subtraction. Data presented on these graphs are the averages of nine observations ± standard error (SE). There were qualitative differences in the cDNA–DNA melt temperatures of psbI and the negative control, psbA , at the different incubation temperatures but these differences were statistically insignificant. Different pH also had no significant effect on the cDNA–DNA melt temperatures and two-way analysis of variance detected no interaction between the temperature and pH for either gene; ( B ) RNase H-dependent PCR (rhPCR) was used in an attempt to detect single base changes between the psbI gDNA and cDNA extracted from Chara incubated in different temperatures. This assay was designed to suppress PCR amplification of the genomic sequence but not cDNA made from an edited transcript. The quantitative PCR (qPCR) amplification curves show that gDNA and cDNA amplified with sigmoidal curves with standard PCR but that both gDNA and cDNA were suppressed by the addition of an rhPCR primer despite the presence of RNase H2 in the reactions. This suggests no editing occurred at any of the tested temperatures; ( C ) cDNAs were PCR amplified and sequenced from total RNA extracted from Chara grown at 20 °C, 25 °C, and 30 °C. The sequences all matched the genomic sequence, suggesting no editing had taken place.
Figure Legend Snippet: Three follow-up experiments attempting to detect three putative RNA editing sites in the C. vulgaris psbI mRNA. ( A ) Chara was grown in nine different environmental conditions: 20 °C, 25 °C, 30 °C and pH 6, 7, 8, in triplicate, and then DNA + RNA were extracted from each. The melt temperatures for genomic DNA (gDNA) and complementary DNA (cDNA) were determined as paired reactions using high resolution melt analysis. Melt temperature differences between gDNA and cDNA derived amplicons were calculated by subtraction. Data presented on these graphs are the averages of nine observations ± standard error (SE). There were qualitative differences in the cDNA–DNA melt temperatures of psbI and the negative control, psbA , at the different incubation temperatures but these differences were statistically insignificant. Different pH also had no significant effect on the cDNA–DNA melt temperatures and two-way analysis of variance detected no interaction between the temperature and pH for either gene; ( B ) RNase H-dependent PCR (rhPCR) was used in an attempt to detect single base changes between the psbI gDNA and cDNA extracted from Chara incubated in different temperatures. This assay was designed to suppress PCR amplification of the genomic sequence but not cDNA made from an edited transcript. The quantitative PCR (qPCR) amplification curves show that gDNA and cDNA amplified with sigmoidal curves with standard PCR but that both gDNA and cDNA were suppressed by the addition of an rhPCR primer despite the presence of RNase H2 in the reactions. This suggests no editing occurred at any of the tested temperatures; ( C ) cDNAs were PCR amplified and sequenced from total RNA extracted from Chara grown at 20 °C, 25 °C, and 30 °C. The sequences all matched the genomic sequence, suggesting no editing had taken place.

Techniques Used: Derivative Assay, Negative Control, Incubation, RNase H-dependent PCR, Polymerase Chain Reaction, Amplification, Sequencing, Real-time Polymerase Chain Reaction

3) Product Images from "Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis"

Article Title: Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis

Journal: Genes

doi: 10.3390/genes8020050

S. dysenteriae produces both RyA1 and RyfA2. ( a ) Northern blot analysis using total RNA isolated from wild-type S. dysenteriae following growth to the mid-logarithmic phase at 37 °C and a radio-labeled probe specific to sequences conserved between RyfA1 and RyfA2. The predicted sizes of RyfA1 and RyfA2 are 303 and 305 nt, respectively. ( b ) Schematic depicting the location and sequence specificity of the amplification primers used in the reverse transcriptase analysis. Each forward primer overlaps the five-nucleotide variable region of RyfA1 or RyfA2, thus providing specificity of amplification. ( c ) Reverse transcriptase PCR demonstrating that both RyfA1 and RyfA2 are produced by wild-type S. dysenteriae under the conditions tested. Using the ryfA1 specific forward primer, amplification occurs when complementary DNA (cDNA) generated from S. dysenteriae or a plasmid carrying ryfA1 is used as a template (pRyfA1), but not when a plasmid carrying ryfA2 is used as a template (pRyfA2). Similarly, using the ryfA2 specific forward primer, amplification occurs when cDNA generated from S. dysenteriae or a plasmid carrying ryfA2 is used as a template, but not when a plasmid carrying ryfA2 is used as a template. The RNA used to generate the cDNA amplification template is used itself as a template with each primer set to ensure that the sample is free from DNA contamination.
Figure Legend Snippet: S. dysenteriae produces both RyA1 and RyfA2. ( a ) Northern blot analysis using total RNA isolated from wild-type S. dysenteriae following growth to the mid-logarithmic phase at 37 °C and a radio-labeled probe specific to sequences conserved between RyfA1 and RyfA2. The predicted sizes of RyfA1 and RyfA2 are 303 and 305 nt, respectively. ( b ) Schematic depicting the location and sequence specificity of the amplification primers used in the reverse transcriptase analysis. Each forward primer overlaps the five-nucleotide variable region of RyfA1 or RyfA2, thus providing specificity of amplification. ( c ) Reverse transcriptase PCR demonstrating that both RyfA1 and RyfA2 are produced by wild-type S. dysenteriae under the conditions tested. Using the ryfA1 specific forward primer, amplification occurs when complementary DNA (cDNA) generated from S. dysenteriae or a plasmid carrying ryfA1 is used as a template (pRyfA1), but not when a plasmid carrying ryfA2 is used as a template (pRyfA2). Similarly, using the ryfA2 specific forward primer, amplification occurs when cDNA generated from S. dysenteriae or a plasmid carrying ryfA2 is used as a template, but not when a plasmid carrying ryfA2 is used as a template. The RNA used to generate the cDNA amplification template is used itself as a template with each primer set to ensure that the sample is free from DNA contamination.

Techniques Used: Northern Blot, Isolation, Labeling, Sequencing, Amplification, Polymerase Chain Reaction, Produced, Generated, Plasmid Preparation

4) Product Images from "Expression of Coagulation-Related Protein Genes During LPS-Induced Preterm Delivery in the Pregnant Mouse"

Article Title: Expression of Coagulation-Related Protein Genes During LPS-Induced Preterm Delivery in the Pregnant Mouse

Journal: Reproductive Sciences

doi: 10.1177/1933719111404607

Representative DNA gels demonstrating the cDNA amplicons for PAI-1 , TF , Par1 , Par2 , Fgl2 , and TM . All of these amplicons were sequence verified to confirm their identity as the target mouse genes. PAI-1 indicates plasminogen activator inhibitor 1; TF
Figure Legend Snippet: Representative DNA gels demonstrating the cDNA amplicons for PAI-1 , TF , Par1 , Par2 , Fgl2 , and TM . All of these amplicons were sequence verified to confirm their identity as the target mouse genes. PAI-1 indicates plasminogen activator inhibitor 1; TF

Techniques Used: Sequencing

5) Product Images from "Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos"

Article Title: Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos

Journal: Scientific Reports

doi: 10.1038/s41598-018-26345-0

Copy numbers per oocyte or embryo of Gdf9 , Pou5f1 , and Nanog mRNAs were quantified by Localize analysis of RNA-FISH assays or droplet digital PCR (ddPCR) analysis of cDNA. ( A ) The average number of Gdf9 , Pou5f1 , and Nanog transcripts in cumulus oocyte complexes (COCs, black bars), MII-oocytes (white bars), 1-cell embryos (light grey bars) and 2-cell embryos (dark grey bars) was determined based on ddPCR (left side) or RNA-FISH (right side). Each ddPCR reaction was performed using 3–4 biological replicates of cDNA with each replicate generated from a pool of 15–20 oocytes or embryos. The number of each transcript in each oocyte or embryo (y-axis) was calculated as described in Methods. Standard error of the mean (SEM) within each experimental group is shown. One pg of gBlock synthetic DNA for each candidate gene served as the positive control for each ddPCR assay. ( B ) The average number of Gdf9 , Pou5f1 , and Nano g transcripts (±SEM) detected by RNA-FISH and counted by Localize in individual GV-oocytes (black bar), MII-oocytes (white bar), 1-cell embryos (light grey bar), and 2-cell embryos (dark grey bars) are shown. The number (n) of oocytes and embryos analyzed for each transcript is indicated in each bar. One-way ANOVA and Tukey pair-wise comparison post-test analyses were performed to compare the abundance of each mRNA in each oocyte and embryo stage. Statistical significance was defined as P
Figure Legend Snippet: Copy numbers per oocyte or embryo of Gdf9 , Pou5f1 , and Nanog mRNAs were quantified by Localize analysis of RNA-FISH assays or droplet digital PCR (ddPCR) analysis of cDNA. ( A ) The average number of Gdf9 , Pou5f1 , and Nanog transcripts in cumulus oocyte complexes (COCs, black bars), MII-oocytes (white bars), 1-cell embryos (light grey bars) and 2-cell embryos (dark grey bars) was determined based on ddPCR (left side) or RNA-FISH (right side). Each ddPCR reaction was performed using 3–4 biological replicates of cDNA with each replicate generated from a pool of 15–20 oocytes or embryos. The number of each transcript in each oocyte or embryo (y-axis) was calculated as described in Methods. Standard error of the mean (SEM) within each experimental group is shown. One pg of gBlock synthetic DNA for each candidate gene served as the positive control for each ddPCR assay. ( B ) The average number of Gdf9 , Pou5f1 , and Nano g transcripts (±SEM) detected by RNA-FISH and counted by Localize in individual GV-oocytes (black bar), MII-oocytes (white bar), 1-cell embryos (light grey bar), and 2-cell embryos (dark grey bars) are shown. The number (n) of oocytes and embryos analyzed for each transcript is indicated in each bar. One-way ANOVA and Tukey pair-wise comparison post-test analyses were performed to compare the abundance of each mRNA in each oocyte and embryo stage. Statistical significance was defined as P

Techniques Used: Fluorescence In Situ Hybridization, Digital PCR, Generated, Positive Control

Related Articles

SYBR Green Assay:

Article Title: Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos
Article Snippet: .. Complementary DNA (cDNA) from oocytes and embryos were diluted 2-fold prior to combination with 1XQX200 ddPCR Evagreen Supermix (BioRad Laboratories), which includes a proprietary SYBR green fluorescent dye and RNA polymerase, and 100 μM of gene specific primers (Table ). .. In addition, primers were combined with either no template (PCR negative control) or synthetic-produced gBlocks Gene Fragments (10 pg/μL, Integrated DNA Technology, Table ) which represented the PCR positive control.

Article Title: Adipocyte OGT governs diet-induced hyperphagia and obesity
Article Snippet: .. Complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). cDNA was amplified and analyzed using iQ SYBR Green Supermix (Bio-Rad) and the LightCycler 480 Instrument II (Roche) as described previously . ..

Amplification:

Article Title: Adipocyte OGT governs diet-induced hyperphagia and obesity
Article Snippet: .. Complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). cDNA was amplified and analyzed using iQ SYBR Green Supermix (Bio-Rad) and the LightCycler 480 Instrument II (Roche) as described previously . ..

Synthesized:

Article Title: Fluid resuscitation with lactated Ringer’s solution vs normal saline in acute pancreatitis: A triple-blind, randomized, controlled trial
Article Snippet: .. Complementary DNA (cDNA) was synthesized from 1 µg RNA sample using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). .. Subsequent qPCR was performed in a DNA Engine, Peltier Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) using iTaq™ Universal SYBR® Green Super mix and the corresponding primers: GAPDH forward: 5’-CTGTGTCTTTCCGCTGTTTTC-3’ and reverse: 5’-TGTGCTGTGCTTATGGTCTCA-3’; IL-1β forward: 5’-AAAAATGCCTCGTGCTGTCT-3’ and reverse: 5’-TCGTTGCTTGTCTCTCCTTG-3’; tumor necrosis factor alpha (TNFα) forward: 5’-AACTCCCAGAAAAGCAAGCA-3’ and reverse: 5’-CGAGCAGGAATGAGAAGAGG-3’; mannose receptor, C type 1 (MRC1) forward: 5’-GGATGGATGGCTCTGGTG-3’ and reverse: 5’-TCTGGTAGGAAACGCTGGT-3’.

Article Title: Expression of Coagulation-Related Protein Genes During LPS-Induced Preterm Delivery in the Pregnant Mouse
Article Snippet: .. For quantitative analysis of mRNA expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), complementary DNA (cDNA) was synthesized from 1 μg samples of RNA template using the iScript cDNA Synthesis kit (Bio-Rad Laboratories) with a mix of oligo-dT and random sequence primers. .. Next, the PCR was performed using the iTaq DNA polymerase kit (Bio-Rad Laboratories) and mouse-specific sense and antisense primers for PAI-1 , TF , Par1 , Par2 , Fgl2 , and TM genes (see for primer sequences).

Article Title: Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth, but not pathogenicity, in Sclerotinia sclerotiorum
Article Snippet: .. Complementary DNA (cDNA) was synthesized with 200 ng of template RNA by an iScript cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA) as recommended by the manufacturer. .. To verify the presence and investigate the expression levels of SCD1 and THR1 , primers were designed using a strategy such that the 1.3‐kb fragment of SCD1 would be amplified using the primer pair SCD1F/SCD1R, whereas the 1.1‐kb fragment of THR1 would be amplified using the primer pair THR1F/THR1R.

Article Title: Adipocyte OGT governs diet-induced hyperphagia and obesity
Article Snippet: .. Complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). cDNA was amplified and analyzed using iQ SYBR Green Supermix (Bio-Rad) and the LightCycler 480 Instrument II (Roche) as described previously . ..

Quantitative RT-PCR:

Article Title: Expression of Coagulation-Related Protein Genes During LPS-Induced Preterm Delivery in the Pregnant Mouse
Article Snippet: .. For quantitative analysis of mRNA expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), complementary DNA (cDNA) was synthesized from 1 μg samples of RNA template using the iScript cDNA Synthesis kit (Bio-Rad Laboratories) with a mix of oligo-dT and random sequence primers. .. Next, the PCR was performed using the iTaq DNA polymerase kit (Bio-Rad Laboratories) and mouse-specific sense and antisense primers for PAI-1 , TF , Par1 , Par2 , Fgl2 , and TM genes (see for primer sequences).

Real-time Polymerase Chain Reaction:

Article Title: Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis
Article Snippet: .. To generate complementary DNA (cDNA) for analysis, iScript (Bio-Rad) was used following the manufacturer’s instructions. cDNA samples were diluted 1 to 10 and used as templates in real-time PCR analyses with TaqMan probe chemistries for analysis of ryfA1 , ryfA2, and ryfB2 , or Sybrgreen and iQ Supermix (Bio-Rad) for analysis of ompC , as per the instructions. .. All samples were amplified and analyzed in a Bio-Rad C1000™ Thermal Cycler with a CFX96 Real-Time System under standard reaction conditions optimized for each primer set ( ).

Polymerase Chain Reaction:

Article Title: Expression of Coagulation-Related Protein Genes During LPS-Induced Preterm Delivery in the Pregnant Mouse
Article Snippet: .. For quantitative analysis of mRNA expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), complementary DNA (cDNA) was synthesized from 1 μg samples of RNA template using the iScript cDNA Synthesis kit (Bio-Rad Laboratories) with a mix of oligo-dT and random sequence primers. .. Next, the PCR was performed using the iTaq DNA polymerase kit (Bio-Rad Laboratories) and mouse-specific sense and antisense primers for PAI-1 , TF , Par1 , Par2 , Fgl2 , and TM genes (see for primer sequences).

Expressing:

Article Title: Expression of Coagulation-Related Protein Genes During LPS-Induced Preterm Delivery in the Pregnant Mouse
Article Snippet: .. For quantitative analysis of mRNA expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), complementary DNA (cDNA) was synthesized from 1 μg samples of RNA template using the iScript cDNA Synthesis kit (Bio-Rad Laboratories) with a mix of oligo-dT and random sequence primers. .. Next, the PCR was performed using the iTaq DNA polymerase kit (Bio-Rad Laboratories) and mouse-specific sense and antisense primers for PAI-1 , TF , Par1 , Par2 , Fgl2 , and TM genes (see for primer sequences).

Sequencing:

Article Title: Expression of Coagulation-Related Protein Genes During LPS-Induced Preterm Delivery in the Pregnant Mouse
Article Snippet: .. For quantitative analysis of mRNA expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), complementary DNA (cDNA) was synthesized from 1 μg samples of RNA template using the iScript cDNA Synthesis kit (Bio-Rad Laboratories) with a mix of oligo-dT and random sequence primers. .. Next, the PCR was performed using the iTaq DNA polymerase kit (Bio-Rad Laboratories) and mouse-specific sense and antisense primers for PAI-1 , TF , Par1 , Par2 , Fgl2 , and TM genes (see for primer sequences).

Produced:

Article Title: Deep Transcriptome Sequencing of Two Green Algae, Chara vulgaris and Chlamydomonas reinhardtii, Provides No Evidence of Organellar RNA Editing
Article Snippet: .. Complementary DNA (cDNA) was produced from DNase treated RNA using Bio-Rad’s iScript cDNA synthesis kit (Hercules, CA, USA). .. Amplicons were produced as paired PCR reactions, one genomic DNA (gDNA) for each cDNA, harvested from the nine pH + temperature conditions using Bio-Rad’s Precision Melt Supermix with 25 ng of gDNA or cDNA template and 10 pmol of each primer.

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    Bio-Rad flhf cdna
    Genetic organization of flhBAFG and fliA genes, and <t>flhF</t> gene expression at 28°C. A . Genetic organization and transcriptional units of flhBAFG and fliA , with arrows showing the direction of transcription. Recognition sites for Eco RI and Stu I are indicated by E and S, respectively. Vertical bars on the map denote the positions and orientation of the Tn 3 - gusA insertion. The two lines with arrowheads beneath the restriction enzyme map indicate the direction and extent of transcription. <t>cDNA</t> synthesized by reverse transcriptase with primers RT1 and RT2 was analyzed by PCR. Thick bars indicate the six expected PCR products, which were verified by electrophoresis on 1.5% agarose gel. B . Photographs of swim assay plates showing the swimming motility of the BGR1 mutant fliA ::Tn 3 - gusA45 and complementation with pBGFA carrying the fliA gene at 28°C and 37°C. C . Expression levels of the flhF gene in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9, and the flhC mutant BGF41 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments.
    Flhf Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad cdna transcripts
    Effect of diet-reversal on CIDE-A expression . Mice were weaned onto the indicated diet for 33 weeks. The mice were then switched to the indicated diets for an additional 14 weeks. RNA was isolated from frozen liver as described. Real Time <t>RT-PCR</t> was performed on the <t>cDNA</t> transcripts using CIDE-A forward and reverse primers. Housekeeping genes, ACTG (actin gamma cytoplasmic) and GADPH (glyceraldehyde-III phosphate dehydrogenase), were utilized for normalization as described. Relative expression levels were calculated using normalization factors derived from geNorm analysis of ACTG and GADPH using the delta-delta CT method. CIDE-A expression levels in control mice fed standard chow for the entire feeding period (SC-SC) were given a value of 1.0. Changes in CIDE-A expression due to diet were expressed relative to the control mice. N = 4 (for each group). SC, standard chow; HF, high-fat diet.
    Cdna Transcripts, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad semi quantitative rt pcr cdna
    Regulation of M. smegmatis adenylate cyclase genes under starvation conditions. Semi-quantitative <t>RT-PCR</t> was used to compare adenylate cyclase mRNA levels between late-log phase cultures incubated for 4 h in mycomedia (control) or PBS (starvation). (A) A representative depiction of PCR-amplified <t>cDNA</t> separated using agarose gel electrophoresis. (B) Quantification of control (black bars) and starvation (white bars) PCR products depicted in A. (C) Average of three different experiments represented as fold differences of starvation compared to control expression. ∗ indicates statistically significant difference between expression in control and starvation for an individual gene ( P
    Semi Quantitative Rt Pcr Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad cdna fragment levels
    Comparison different PCR methods and impact on quantification. Graphic representation of the quantification of dystrophin <t>cDNA</t> construct templates lacking or containing exon 51 mixed in different ratios ranging from 0% to 100% using nested PCR (40, 50 and 60 cycles), <t>qPCR</t> (LinRegPCR and Pfaffl methods) and ddPCR. The dashed red line indicates the theoretical template ratios. The solid black ddPCR line is almost indistinguishable from the theoretical line and both lines are superimposed. Data is based on one experiment using 2 replicates for ddPCR and 3 replicates for qPCR.
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    Genetic organization of flhBAFG and fliA genes, and flhF gene expression at 28°C. A . Genetic organization and transcriptional units of flhBAFG and fliA , with arrows showing the direction of transcription. Recognition sites for Eco RI and Stu I are indicated by E and S, respectively. Vertical bars on the map denote the positions and orientation of the Tn 3 - gusA insertion. The two lines with arrowheads beneath the restriction enzyme map indicate the direction and extent of transcription. cDNA synthesized by reverse transcriptase with primers RT1 and RT2 was analyzed by PCR. Thick bars indicate the six expected PCR products, which were verified by electrophoresis on 1.5% agarose gel. B . Photographs of swim assay plates showing the swimming motility of the BGR1 mutant fliA ::Tn 3 - gusA45 and complementation with pBGFA carrying the fliA gene at 28°C and 37°C. C . Expression levels of the flhF gene in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9, and the flhC mutant BGF41 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments.

    Journal: PLoS ONE

    Article Title: Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae

    doi: 10.1371/journal.pone.0084831

    Figure Lengend Snippet: Genetic organization of flhBAFG and fliA genes, and flhF gene expression at 28°C. A . Genetic organization and transcriptional units of flhBAFG and fliA , with arrows showing the direction of transcription. Recognition sites for Eco RI and Stu I are indicated by E and S, respectively. Vertical bars on the map denote the positions and orientation of the Tn 3 - gusA insertion. The two lines with arrowheads beneath the restriction enzyme map indicate the direction and extent of transcription. cDNA synthesized by reverse transcriptase with primers RT1 and RT2 was analyzed by PCR. Thick bars indicate the six expected PCR products, which were verified by electrophoresis on 1.5% agarose gel. B . Photographs of swim assay plates showing the swimming motility of the BGR1 mutant fliA ::Tn 3 - gusA45 and complementation with pBGFA carrying the fliA gene at 28°C and 37°C. C . Expression levels of the flhF gene in the wild-type BGR1, the tofI mutant BGS2, and the qsmR mutant BGS9, and the flhC mutant BGF41 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments.

    Article Snippet: For qRT-PCR analysis, the primers of flhC-cDNA, flhF-cDNA, flhC-qRTF, flhC-qRTR, flhF-qRTF, and flhF-qRTR were used ( ). qRT-PCR analysis was performed according to the manufacturer's instructions except using SsoFast™ EvaGreen Supermix (Bio-Rad) with 25 ng of cDNA as template. qRT-PCR was performed using a thermal cycler (Model C1000™; Bio-Rad) under the following conditions: 98°C for 2 min, followed by 35 cycles at 98°C for 20 s, 65°C for 30 s, and 72°C for 30 s. The 16S rRNA gene was used for data normalization.

    Techniques: Expressing, Synthesized, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Mutagenesis, Quantitative RT-PCR

    Effect of diet-reversal on CIDE-A expression . Mice were weaned onto the indicated diet for 33 weeks. The mice were then switched to the indicated diets for an additional 14 weeks. RNA was isolated from frozen liver as described. Real Time RT-PCR was performed on the cDNA transcripts using CIDE-A forward and reverse primers. Housekeeping genes, ACTG (actin gamma cytoplasmic) and GADPH (glyceraldehyde-III phosphate dehydrogenase), were utilized for normalization as described. Relative expression levels were calculated using normalization factors derived from geNorm analysis of ACTG and GADPH using the delta-delta CT method. CIDE-A expression levels in control mice fed standard chow for the entire feeding period (SC-SC) were given a value of 1.0. Changes in CIDE-A expression due to diet were expressed relative to the control mice. N = 4 (for each group). SC, standard chow; HF, high-fat diet.

    Journal: Comparative Hepatology

    Article Title: CIDE-A is expressed in liver of old mice and in type 2 diabetic mouse liver exhibiting steatosis

    doi: 10.1186/1476-5926-6-4

    Figure Lengend Snippet: Effect of diet-reversal on CIDE-A expression . Mice were weaned onto the indicated diet for 33 weeks. The mice were then switched to the indicated diets for an additional 14 weeks. RNA was isolated from frozen liver as described. Real Time RT-PCR was performed on the cDNA transcripts using CIDE-A forward and reverse primers. Housekeeping genes, ACTG (actin gamma cytoplasmic) and GADPH (glyceraldehyde-III phosphate dehydrogenase), were utilized for normalization as described. Relative expression levels were calculated using normalization factors derived from geNorm analysis of ACTG and GADPH using the delta-delta CT method. CIDE-A expression levels in control mice fed standard chow for the entire feeding period (SC-SC) were given a value of 1.0. Changes in CIDE-A expression due to diet were expressed relative to the control mice. N = 4 (for each group). SC, standard chow; HF, high-fat diet.

    Article Snippet: Real-time RT-PCR RNA was isolated from frozen liver as described above. cDNA transcripts were created using iScript cDNA Synthesis Kit (Cat# 170-8891, Bio-Rad Laboratories, Hercules, CA).

    Techniques: Expressing, Mouse Assay, Isolation, Quantitative RT-PCR, Derivative Assay

    CIDE-A Northern and Immunoblot analyses . A) Northern analysis of RNA extracted from normal (C) and type 2 diabetic (D) liver and heart tissue. Total RNA (10 μg) from the appropriate tissues was resolved by denaturing agarose gel electrophoresis, transferred to positively charged nylon membrane, hybridized with the [α- 32 P]dCTP-labeled mouse CIDE-A cDNA and exposed to Bio-Max MR film. Ethidium bromide stain of RNA (10 μg/lane) prior to transfer to nylon membrane. The values represent the level of CIDE-A gene expression for the individual tissue as determined by DNA microarray analysis (ND: not determined). The approximated size (1.3 kb) of the CIDE-A mRNA is noted on the right. B) Immunoblot demonstrating increased CIDE-A protein levels in type 2 diabetic mouse liver. Sixty μg of liver and heart extract was electrophoresed on a 12.5% SDS-polyacrylamide gel and the resolved proteins transferred to a nitrocellulose membrane. The membrane was immunoblotted using a rabbit anti-mouse CIDE-A polyclonal antibody and a goat anti-rabbit IgG polyclonal antibody conjugated to horseradish peroxidase. Arrow indicates mouse CIDE-A. The values represent the level of CIDE-A gene expression for the individual tissue as determined by DNA microarray analysis (ND: not determined).

    Journal: Comparative Hepatology

    Article Title: CIDE-A is expressed in liver of old mice and in type 2 diabetic mouse liver exhibiting steatosis

    doi: 10.1186/1476-5926-6-4

    Figure Lengend Snippet: CIDE-A Northern and Immunoblot analyses . A) Northern analysis of RNA extracted from normal (C) and type 2 diabetic (D) liver and heart tissue. Total RNA (10 μg) from the appropriate tissues was resolved by denaturing agarose gel electrophoresis, transferred to positively charged nylon membrane, hybridized with the [α- 32 P]dCTP-labeled mouse CIDE-A cDNA and exposed to Bio-Max MR film. Ethidium bromide stain of RNA (10 μg/lane) prior to transfer to nylon membrane. The values represent the level of CIDE-A gene expression for the individual tissue as determined by DNA microarray analysis (ND: not determined). The approximated size (1.3 kb) of the CIDE-A mRNA is noted on the right. B) Immunoblot demonstrating increased CIDE-A protein levels in type 2 diabetic mouse liver. Sixty μg of liver and heart extract was electrophoresed on a 12.5% SDS-polyacrylamide gel and the resolved proteins transferred to a nitrocellulose membrane. The membrane was immunoblotted using a rabbit anti-mouse CIDE-A polyclonal antibody and a goat anti-rabbit IgG polyclonal antibody conjugated to horseradish peroxidase. Arrow indicates mouse CIDE-A. The values represent the level of CIDE-A gene expression for the individual tissue as determined by DNA microarray analysis (ND: not determined).

    Article Snippet: Real-time RT-PCR RNA was isolated from frozen liver as described above. cDNA transcripts were created using iScript cDNA Synthesis Kit (Cat# 170-8891, Bio-Rad Laboratories, Hercules, CA).

    Techniques: Northern Blot, Agarose Gel Electrophoresis, Labeling, Staining, Expressing, Microarray

    Regulation of M. smegmatis adenylate cyclase genes under starvation conditions. Semi-quantitative RT-PCR was used to compare adenylate cyclase mRNA levels between late-log phase cultures incubated for 4 h in mycomedia (control) or PBS (starvation). (A) A representative depiction of PCR-amplified cDNA separated using agarose gel electrophoresis. (B) Quantification of control (black bars) and starvation (white bars) PCR products depicted in A. (C) Average of three different experiments represented as fold differences of starvation compared to control expression. ∗ indicates statistically significant difference between expression in control and starvation for an individual gene ( P

    Journal: PeerJ

    Article Title: The role of transcriptional regulation in maintaining the availability of mycobacterial adenylate cyclases

    doi: 10.7717/peerj.298

    Figure Lengend Snippet: Regulation of M. smegmatis adenylate cyclase genes under starvation conditions. Semi-quantitative RT-PCR was used to compare adenylate cyclase mRNA levels between late-log phase cultures incubated for 4 h in mycomedia (control) or PBS (starvation). (A) A representative depiction of PCR-amplified cDNA separated using agarose gel electrophoresis. (B) Quantification of control (black bars) and starvation (white bars) PCR products depicted in A. (C) Average of three different experiments represented as fold differences of starvation compared to control expression. ∗ indicates statistically significant difference between expression in control and starvation for an individual gene ( P

    Article Snippet: Semi-quantitative RT-PCR cDNA was prepared from 0.5 µg of RNA using the iScript cDNA synthesis kit according to the manufacture’s specifications (BioRad).

    Techniques: Quantitative RT-PCR, Incubation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Expressing

    Comparison different PCR methods and impact on quantification. Graphic representation of the quantification of dystrophin cDNA construct templates lacking or containing exon 51 mixed in different ratios ranging from 0% to 100% using nested PCR (40, 50 and 60 cycles), qPCR (LinRegPCR and Pfaffl methods) and ddPCR. The dashed red line indicates the theoretical template ratios. The solid black ddPCR line is almost indistinguishable from the theoretical line and both lines are superimposed. Data is based on one experiment using 2 replicates for ddPCR and 3 replicates for qPCR.

    Journal: PLoS ONE

    Article Title: Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy

    doi: 10.1371/journal.pone.0162467

    Figure Lengend Snippet: Comparison different PCR methods and impact on quantification. Graphic representation of the quantification of dystrophin cDNA construct templates lacking or containing exon 51 mixed in different ratios ranging from 0% to 100% using nested PCR (40, 50 and 60 cycles), qPCR (LinRegPCR and Pfaffl methods) and ddPCR. The dashed red line indicates the theoretical template ratios. The solid black ddPCR line is almost indistinguishable from the theoretical line and both lines are superimposed. Data is based on one experiment using 2 replicates for ddPCR and 3 replicates for qPCR.

    Article Snippet: In each qPCR run, a no template control (NTC) was included as negative control. cDNA fragment levels were determined on a CFX96 real-time thermal cycler (BioRad) using the following protocol: 95°C for 10 min, then 40 cycles of 95°C for 15 sec and 60°C for 1 min. qPCR data was analyzed using both the Pfaffl method [ ] and the LinRegPCR method [ ].

    Techniques: Polymerase Chain Reaction, Construct, Nested PCR, Real-time Polymerase Chain Reaction