Journal: Genome Biology

Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis

doi: 10.1186/s13059-015-0629-x

Figure Lengend Snippet: Extensive mRNA isoform loss and intron retention in RRMS. ( A ) Total exon loss and intron gain events across the genome in RRMS (N = 6) and CTRL (N = 8) were determined from RNA-seq analysis (see Methods ). Results are expressed as average normalized counts or reads across known exons and introns in CTRL and RRMS. ( B ) Percent abundance of transcript alterations in RRMS relative to CTRL PBMC determined from analysis of genome-wide RNA-seq data. ( C ) Example of intron retention in mRNA encoded by MBP . Green arrows identify exons. Red circles identify retention of intron sequences in mature mRNA in RRMS. Orange and blue arrows identify two isoforms. ( D , E ) Examples of 5’ mRNA shortening, CSF1R , and 3’ mRNA shortening, NFATC1 in RRMS. Red arrows identify loss of exons in RRMS and green arrows identify exons expressed at equal levels between CTRL and RRMS. ( F ) Expression levels of individual exons and the MBP intron in CTRL (N = 12) and RRMS (N = 12) was determined by quantitative PCR and normalized to CTRL = 1.0 after normalization to transcript levels of GAPDH , error bars are S.D. * = P

Article Snippet: Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

Techniques: RNA Sequencing Assay, Genome Wide, Expressing, Real-time Polymerase Chain Reaction

Journal: Genome Biology

Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis

doi: 10.1186/s13059-015-0629-x

Figure Lengend Snippet: Loss of TROVE2 and/or SSB via small RNA interference increases poly(A) + Y1 RNA, rRNAs, and U1 RNA, misprocessed 18S and 28S rRNA, and alters mRNA length. ( A ) Selective siRNA-mediated knockdown of TROVE2 or SSB in THP-1 cells or Jurkat T cells. Transcript levels of TROVE2 and SSB were determined by quantitative PCR using oligo d(T) for cDNA synthesis. Results are normalized to cells transfected with a non-specific scrambled siRNA negative control after normalization to transcript levels of GAPDH . ( B ) Poly(A) + Y1 RNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis. Results normalized to CTRL (transfection of scrambled siRNA). ( C ) As in ( B ) except levels of 18S and 28S rRNAs were determined. ( D ) As in (B) except levels of poly(A) + U1 snRNA were determined. ( E , F ) Transcript levels of misprocessed rRNAs in THP-1 (E) and Jurkat T cells (F) (-90, -60, -30 bp) were determined and are expressed relative to cells transfected with a scrambled siRNA as described in Figure 2 . ( G ) PCR strategy to test altered lengths of MBP , CSF1R , and NFATC1 transcripts. ( H ) As in (A) except transcript levels of the MBP intron, CSF1R exons, and NFATC1 short to long mRNA isoform ratios were determined by quantitative PCR and normalized to CTRL. Each experiment was performed a minimum of three times, results are expressed as mean ± S.D * P

Article Snippet: Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Negative Control, Polymerase Chain Reaction

Journal: Genome Biology

Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis

doi: 10.1186/s13059-015-0629-x

Figure Lengend Snippet: Increased total and polyadenylated U snRNAs in RRMS. ( A ) Total U1 RNA transcript levels were determined by quantitative PCR using random hexamers for cDNA synthesis in CTRL (N = 24) and RRMS (N = 22). Individual transcripts were normalized to GAPDH transcript levels. ( B ) As in (A) except total poly(A) + U1 snRNA levels were determined by quantitative PCR using oligo-dT for cDNA synthesis in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), SLE (N = 24), RA (N = 18), and NMO (N = 22). ( C ) As in (A), except transcript levels were determined by whole genome RNA-sequencing (RNA-seq) and normalized to CTRL = 1; CTRL (N = 8) and RRMS (N = 6). ( D ) As in (A, B) except transcript levels of U11 and U12 snRNAs were determined by quantitative PCR using oligo d(T) for poly(A) + or random hexamer primers for total U11 or U12 snRNA.

Article Snippet: Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

Techniques: Real-time Polymerase Chain Reaction, Mass Spectrometry, RNA Sequencing Assay, Random Hexamer Labeling

Journal: Genome Biology

Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis

doi: 10.1186/s13059-015-0629-x

Figure Lengend Snippet: Ro60 and La proteins are depressed in RRMS. ( A ) Ro60 ( TROVE2 ) and La ( SSB ) transcript levels in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), RA (N = 18), SLE (N = 24), NMO (N = 22), and PD (N = 19) were determined by quantitative PCR after cDNA synthesis using oligo-dT. Results are normalized to CTRL = 1.0 after normalization to transcript levels of GAPDH , error bars are S.D. ( B ) As in (A) using whole genome RNA-sequencing data. ( C ) Western blotting to determine Ro60 and La protein levels in PBMC from CTRL (N = 9) and RRMS (N = 8). ( D ) Quantitative estimates of protein abundance relative to β-actin. * P

Article Snippet: Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

Techniques: Mass Spectrometry, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Western Blot

Journal: Genome Biology

Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis

doi: 10.1186/s13059-015-0629-x

Figure Lengend Snippet: Interferon-β1b (IFN-β1b) therapy corrects aberrant levels of TROVE2 , SSB , poly (A) + Y1 RNA, and poly(A) + U1 snRNA in RRMS. Blood samples were collected in PaxGene tubes from CTRL (N = 12), RRMS subjects not on IFN-β1b (RRMS - IFN-β1b, N = 12), and RRMS subjects on stable IFN-β1b therapy (RRMS + IFN-β1b, N = 4). Oligo dT was used for cDNA synthesis. Transcript levels of TROVE2 and SSB ( A ) or poly(A) + Y1 RNA and poly(A) + U1 RNA ( B ) were determined by quantitative PCR and normalized to CTRL = 1 after normalization to levels of GAPDH . Error bars are ± S.D. * P

Article Snippet: Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

Techniques: Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

doi: 10.1038/srep26616

Figure Lengend Snippet: Restriction of HBV by SAMHD1 is dependent upon dNTPase activity, is inhibited by phosphorylation of T592 and is rescued by addition of deoxynucleosides. ( a , b ) HepG2.2.15 cells were transfected with 1 μg of plasmids coding for wild-type or mutant FLAG-tagged SAMHD1 or with 1 μg of empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements for 48 hours. ( a ) Detection of overexpressed FLAG-SAMHD1 by Western blotting. Detection of actin was used as a loading control. ( b ) HBV DNA from the supernatant was quantified by qPCR. The mean ± SEM of technical triplicates from one representative experiment out of three independent experiments is represented. ( c ) HepG2 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements to arrest the cell cycle. When indicated, 10 mM of hydroxyurea (HU) were added at the time of medium change. Quantification of dATP levels 3 days post medium change were determined by single-nucleotide incorporation assay. The mean ± SEM of technical duplicates is represented. ( d ) HepG2.2.15 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or with an empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements. When indicated, deoxynucleosides (dNs, 2 mM each) or solvent were added at the time of medium change. HBV DNA from the supernatant was quantified by qPCR 3 days post medium change. For each independent experiment, fold-changes to empty vector were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is represented. ( b , d ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (**p

Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

Techniques: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

doi: 10.1038/srep26616

Figure Lengend Snippet: Overexpression of SAMHD1 reduces extracellular and intracellular levels of HBV DNA in HepG2.2.15 cells without affecting viral RNA. HepG2.2.15 cells were transfected with ( a ) increasing amounts or ( b – d ) 1 μg of a SAMHD1-coding plasmid. The total amount of plasmid DNA was adjusted to 1 μg by addition of empty vector. Twenty-four hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and further cultured in serum-free medium without supplements. Forty-eight hours later, HBV DNA from the supernatant ( a , d ) and intracellular HBV DNA from cytoplasmic fractions ( c ) were quantified by qPCR. ( b ) The levels of intracellular HBV RNAs were determined by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( a ) The mean ± SEM of the technical triplicates is shown. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

doi: 10.1038/srep26616

Figure Lengend Snippet: dATP levels are affected by HBV infection. SAMHD1 is de-phosphorylated at T592 in primary human hepatocytes, and is not affected by HBV infection. ( a ) HepG2-NTCP- control-shRNA cells were infected or not with HBV in presence of 2.5%DMSO and dATP content was measured 4 days post infection. The mean ± SEM of technical duplicates is represented. ( b , c ) Primary human hepatocytes (PHH) from one donnor were infected or not with HBV for 8 or 24 hours. ( b ) SAMHD1 phosphorylation on T592, total SAMHD1, cyclin B1 and GAPDH were detected by Western blotting. Cycling or serum starved HepG2.2.15-control-shRNA cells were used respectively as positive and negative controls for pT592-SAMHD1 and cyclin B1. A representative experiment out of two technical replicates is represented. ( c ) Levels of intracellular HBV RNAs from infected PHH used in ( b ) were determined by RT-qPCR using HBV primer set 3 (see Fig. 2b and Table 1 ; amplified HBV RNA species: 3.5 kb, 2.4 kb and 2.1 kb RNAs). B.d.: below detection. The mean ± SEM of technical triplicates is represented.

Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

Techniques: Infection, shRNA, Western Blot, Quantitative RT-PCR, Amplification

Journal: Scientific Reports

Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

doi: 10.1038/srep26616

Figure Lengend Snippet: SAMHD1 restricts HBV replication in infected HepG2-NTCP cells. Detection of ( a ) SAMHD1 or ( b ) NTCP in SAMHD1 knockdown or control HepG2-NTCP cells by Western blotting. Actin or GAPDH were used as loading controls. ( c ) Equal cell growth and viability was assessed was assessed using ATPLite. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( d ) Control shRNA cells were cultured for 10 days in complete medium supplemented (+) or not (−) with 2.5% DMSO. When indicated cells were infected with HBV 48 hours after start of DMSO treatment. Phosphorylation at residue T592 in SAMHD1, as well as expression of total SAMHD1, cyclin B1 and actin, were detected by Western blotting. ( e ) Cells were cultured for 10 days in 2.5% DMSO-containing medium and infected with a single-round luciferase HIV-1 virus. Luciferase activity was detected 24 hours post infection. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( f-i ) Cells were infected with HBV inoculum, and where indicated, the entry inhibitor MyrcludexB (MyrB, 200 nM), or the reverse transcription inhibitor lamivudine (Lami, 0,5 μM) were added. Cells were cultured in medium containing 2.5% DMSO. Ten days post infection, HBV DNA from the supernatant ( f ), intracellular total HBV DNA ( g ) and cccDNA ( h ) were quantified by qPCR. ( i ) The intracellular HBV RNAs levels were determined 10 days post infection by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( f ) For each individual experiment, fold-changes to the untreated control shRNA were calculated based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is depicted. ( g – i ) For each independent experiment, fold-changes to untreated control shRNA were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of at least 3 independent experiments is represented. ( f – i ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

Techniques: Infection, Western Blot, shRNA, Cell Culture, Expressing, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

doi: 10.1038/srep26616

Figure Lengend Snippet: Silencing of SAMHD1 increases HBV replication in resting HepG2.2.15 cells. ( a ) SAMHD1 or actin protein levels were detected in cell lysates from SAMHD1 knockdown or control HepG2.2.15 cells by Western blotting. ( b ) Phosphorylation on residue T592 of SAMHD1 and cyclin B1 expression were detected by Western blotting of lysates from control shRNA HepG2.2.15 cells cultured for 3 days in complete versus serum-free medium. ( c-e ) HepG2.2.15 cells stably expressing two different SAMHD1 shRNAs or a control shRNA were cultured in serum-free medium for 3 days. ( c ) Luciferase activity (relative luciferase units, RLU) was detected in cells 24 hours post infection with a single-round HIV-1-based lentiviral vector that encoded luciferase. ( d ) Equal cell growth and viability between different shRNA cell lines was assessed using ATPLite. ( e ) The amount of HBV DNA in the supernatant was determined by qPCR and normalized to the control shRNA. In ( c , d ), the mean ± standard error mean (SEM) of three biological replicates of one representative experiment is depicted. In ( e ), the fold-changes to negative control were calculated for each individual experiment based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of four independent experiments is depicted. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

Techniques: Western Blot, Expressing, shRNA, Cell Culture, Stable Transfection, Luciferase, Activity Assay, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Negative Control

Journal: PLoS ONE

Article Title: Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable, but Shows Quantitative Changes during Tumour Progression

doi: 10.1371/journal.pone.0053883

Figure Lengend Snippet: Cloned PCR products from the 5′ (exon 4, italic) and 3′ (exon 16, bold) primer (squared) combination of CD44 in A2058 human melanoma cell line. Direct sequencing shows a CD44 isoform with no v1 or any other variable exons (A) as well as one with truncated v1 (underlined).

Article Snippet: Quantitative PCR Analysis For quantitative measurement of the expressed CD44 variable exons q-PCR reactions were used (iQ SYBR® Green Supermix,Bio-Rad), by cycling conditions 3 min at 95°C, then 40 cycles at 95°C 30 sec, 55°C 30 sec, 72°C 1 min.

Techniques: Clone Assay, Polymerase Chain Reaction, Sequencing

Journal: PLoS ONE

Article Title: Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable, but Shows Quantitative Changes during Tumour Progression

doi: 10.1371/journal.pone.0053883

Figure Lengend Snippet: Relative quantitative expression of CD44 variable exons in cell cultures from metastatic (newborn) and non-metastatic [adult (AP)] human xenograft model (Real-Time PCR measurement) of HT168M1, a human melanoma cell line of originally high variable exon expression level. A. The relative expression level of all variable exons is raised in certain lung metastasis (NM), when remaines low or even decreases in other lung metastases resulting in large error bars. It should be noted that the expression level of the primaries from different localisations [newborn primary (NP), adult primary (AP) and intravenously implanted lung colony (IVLC)] are all comparable, although the slightly higher expression observed in the adult primary is an unexpected finding. B. We used the lung metastasis from the newborn animal with the highest CD44 variable exon expression level (NM = S1T2) for subcutaneous re-implantation into another newborn animal. The expression level in the primary tumour (PNM) and its metastases (MPNM) was 24 times lower on average. C. The liver metastases (LMIVLC) from the intravenously implanted lung colonies (IVLC) showed a decrease in expression, when compared to the lung colonies (IVLC).

Article Snippet: Quantitative PCR Analysis For quantitative measurement of the expressed CD44 variable exons q-PCR reactions were used (iQ SYBR® Green Supermix,Bio-Rad), by cycling conditions 3 min at 95°C, then 40 cycles at 95°C 30 sec, 55°C 30 sec, 72°C 1 min.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable, but Shows Quantitative Changes during Tumour Progression

doi: 10.1371/journal.pone.0053883

Figure Lengend Snippet: Relative quantitative expression of CD44 variable exons in cell cultures from metastatic (newborn) and non-metastatic human xenograft model (Real-Time PCR measurement) of HT199, a human melanoma cell line of originally low variable exon expression level. A. The relative expression level of all variable exons is raised in circulating metastatic cells (NCTC) and metastatic cells (NM) compared to their levels in primary tumours [newborn primary (NP) and adult primary (AP)] and lung colony (IVLC) B. The qualitative fingerprint (bottom line) remains unchanged.

Article Snippet: Quantitative PCR Analysis For quantitative measurement of the expressed CD44 variable exons q-PCR reactions were used (iQ SYBR® Green Supermix,Bio-Rad), by cycling conditions 3 min at 95°C, then 40 cycles at 95°C 30 sec, 55°C 30 sec, 72°C 1 min.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Plant Science

Article Title: Comprehensive Expression Profiling of Rice Tetraspanin Genes Reveals Diverse Roles During Development and Abiotic Stress

doi: 10.3389/fpls.2015.01088

Figure Lengend Snippet: Expression profiling of rice tetraspanin genes in different tissues of rice. (A) Quantitative PCR analysis of transcript levels of rice tetraspanins in tissues namely shoot, root, young leaf (YL), active tillering phase (AT-Phase), stem elongation phase (SE-Phase), spikelets (Spks), young flag leaf (YFL), and mature flag leaf (MFL). For root tissue normalized fold change (log 2 scale) was calculated relative to that in shoot tissue of 7-day-old seedlings. For tissues obtained from field grown plants fold change (log 2 scale) was calculated relative to that in young leaf. (B) Quantitative PCR analysis of transcript levels of rice tetraspanins during progression of senescence in flag leaf of field-grown rice plants. CON: fully expanded MFL; 100% chlorophyll, S1: 80–90% chlorophyll; S2: 60–80% chlorophyll; S3: 40–60% chlorophyll. Normalized fold change (log 2 scale) was calculated relative to that in MFL. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Error bars represent standard error (SE) of three independent biological replicates.

Article Snippet: Quantitative PCR (qPCR) was performed with three biological replicates and two technical replicates using Sso fast Evagreen supermix (Bio-Rad, USA), appropriate primers and Master cycler RealPlex2 (Eppendorf, Germany).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Plant Science

Article Title: Comprehensive Expression Profiling of Rice Tetraspanin Genes Reveals Diverse Roles During Development and Abiotic Stress

doi: 10.3389/fpls.2015.01088

Figure Lengend Snippet: Heat map representing expression profile of rice tetraspanin genes in rice seedlings exposed to nutrient deprivation. Seven-day-old rice seedlings were grown in different nutrient deprivation media such as nitrogen deprivation (N), phosphorous deprivation (P), potassium deprivation (K), sulfur deprivation (S) for different time durations as indicated on the left (in h). Expression levels of tetraspanin genes were determined by quantitative PCR and normalized fold change (log 2 scale) was calculated relative to that in unstressed seedlings. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Hierarchical clustering analysis of relative fold change was performed to prepare a dendrogram and a heat map using Hierarchical Clustering Explorer v3.5 software.

Article Snippet: Quantitative PCR (qPCR) was performed with three biological replicates and two technical replicates using Sso fast Evagreen supermix (Bio-Rad, USA), appropriate primers and Master cycler RealPlex2 (Eppendorf, Germany).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Software

Journal: Frontiers in Plant Science

Article Title: Comprehensive Expression Profiling of Rice Tetraspanin Genes Reveals Diverse Roles During Development and Abiotic Stress

doi: 10.3389/fpls.2015.01088

Figure Lengend Snippet: Heat map representing expression profile of rice tetraspanin genes in rice seedlings exposed to different abiotic stresses. Seven-day-old rice seedlings were exposed to different abiotic stresses such as heat stress at 42°C; salinity stress with 200 mM NaCl; water deficit stress (WDS) imposed by 15% PEG; cold stress at 4°C; oxidative stress with 10 mM H 2 O 2 for different time durations as indicated on the left (duration in h). Expression levels of tetraspanin genes were determined by quantitative PCR and normalized fold change (log 2 scale) was calculated relative to that in unstressed seedlings. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Hierarchical clustering analysis of relative fold change was performed to prepare a dendrogram and a heat map using Hierarchical Clustering Explorer v3.5 software.

Article Snippet: Quantitative PCR (qPCR) was performed with three biological replicates and two technical replicates using Sso fast Evagreen supermix (Bio-Rad, USA), appropriate primers and Master cycler RealPlex2 (Eppendorf, Germany).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Software

Journal: Frontiers in Plant Science

Article Title: Comprehensive Expression Profiling of Rice Tetraspanin Genes Reveals Diverse Roles During Development and Abiotic Stress

doi: 10.3389/fpls.2015.01088

Figure Lengend Snippet: Heat map representing expression profile of rice tetraspanin genes in rice seedlings exposed to different hormones. Seven-day-old seedlings were exposed to exogenous hormones namely abscisic acid (ABA), brassinosteroids (BS), gibberellic acid (GA), and methyl jasmonate (MeJA) for different time durations (in h) as indicated on the left. Expression levels of tetraspanin genes were determined by quantitative PCR and normalized fold change (log 2 scale) was calculated relative to that in untreated seedlings. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Hierarchical clustering analysis of relative fold change was performed to prepare a dendrogram and a heat map using Hierarchical Clustering Explorer v3.5 software.

Article Snippet: Quantitative PCR (qPCR) was performed with three biological replicates and two technical replicates using Sso fast Evagreen supermix (Bio-Rad, USA), appropriate primers and Master cycler RealPlex2 (Eppendorf, Germany).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Software

Journal: Journal of Clinical Microbiology

Article Title: Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

doi: 10.1128/JCM.01764-16

Figure Lengend Snippet: (A) SYBR green dissociation curve analyses of LSG-qPCR amplicons from the Stratagene Mx3000P platform. G1, L . ( V .) braziliensis , L . ( V .) guyanensis , and L . ( V .) panamensis ; G2A/B, L . ( L .) infantum/L . ( L .) chagasi and L . ( L .) tropica ; G3, L . ( L .) amazonensis

Article Snippet: PCRs were run on either of two different qPCR platforms—the Stratagene Mx3000P qPCR system (Stratagene-Agilent) or the ABI 7500 real-time PCR system (ABI Life Technologies)—using the following cycling parameters: 95°C for 15 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. Fluorescence data were collected at the end of each 60°C plateau.

Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: Journal of cancer science & therapy

Article Title: TDP1/TOP1 Ratio as a Promising Indicator for the Response of Small Cell Lung Cancer to Topotecan

doi: 10.4172/1948-5956.1000280

Figure Lengend Snippet: Small cell lung cancer cell lines display good correlation between TDP1 or TOP1 protein level and mRNA cDNA from each of the SCLC lines was generated from 1 μg purified mRNA using the Superscript First Strand RT-PCR and real time PCR analysis was conducted using TDP1 primers (A) or TOP1 primers (C) and an MX3005p qPCR system. The relative quantity of mRNA (RQ value) for each repeat experiment was calculated according to the equation RQ = 2 ΔΔCT and the RQ values normalised to that obtained for the HCC33 cell line. The average of normalised RQ values from 3 independent experiments ± STD is depicted (A C). A graphical representation of the Pearson’s correlation coefficient (r) between SCLC TDP1 protein levels and TDP1 mRNA is shown in (B) and that for SCLC TOP1 protein level and TOP1 mRNA is shown in (D) .

Article Snippet: The mRNA concentration was determined using the NanoDrop 1000 spectrophotometer (Nanodrop, Willmington, USA) at OD 230 nm. cDNA was generated from 1 μg mRNA using the Superscript First Strand RT-PCR system (Invitrogen, Paisley, UK) and real time PCR analysis was carried out on the MX3005p qPCR system (Agilent Technologies, Santa Clara, USA) using Blue qPCR Sybr low ROX mix (Fisher Scientific, Loughborough, UK) for cDNA amplification.

Techniques: Generated, Purification, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Frontiers in Integrative Neuroscience

Article Title: Region- and Cell-Specific Expression of Transmembrane Collagens in Mouse Brain

doi: 10.3389/fnint.2017.00020

Figure Lengend Snippet: Developmental expression of col25a1 in mouse hippocampus. (A) Quantitative RT-PCR (qPCR) shows a developmental increase in col25a1 expression in mouse hippocampus from hippocampal RNA isolated at P0, P7, P15 and P60. Levels of col25a1 are normalized to gapdh . Data are means +/− SEM. **Indicates expression levels differ from levels at P0 at P

Article Snippet: Quantitative real-time PCR (qPCR) was performed on a CFX Connect Real-Time system (BioRad) using iTag SYBRGreen Supermix (BioRad) by the following primers: col25a1 , 5′- GAT TCT CCT CTT CGG CCT CT -3′and 5′- AAA TAA GAA CGG CCA GGG AG -3′; col23a1 , 5′- GCA ATC AGG ACG AGA TGG CT-3′ and 5′- AAA GTC TCC CGG TGT ACC CT-3′; col17a1 , 5′-TGG GAT CAG CTT TGG GCA TC-3′ and 5′- GAC AAA CCA GCG GCT CGG A-3′; col13a1 , 5′- AAG GGA GAA GCA GGC CTA GAG-3′ and 5′-TGG AGT ACC AGG CAA TCC CAG-3′; gapdh , 5′-CGT CCC GTA GAC AAA ATG GT-3′ and 5′-TTG ATG GCA ACA ATC TCC AC-3′.

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Isolation

Journal: Frontiers in Integrative Neuroscience

Article Title: Region- and Cell-Specific Expression of Transmembrane Collagens in Mouse Brain

doi: 10.3389/fnint.2017.00020

Figure Lengend Snippet: Region-specific membrane associated collagens with interrupted triple helices (MACIT) mRNA expression in mouse brain. (A) Quantitative RT-PCR (QPCR) analysis of col13a1, col17a1, col23a1 and col25a1 mRNA levels in different regions of postnatal day 21 (P21) mouse brain and retina. Levels of MACIT mRNA expression are normalized to gapdh in each sample. Data shown represent means +/− standard error of the mean (SEM; error bars). ***Indicates MACIT mRNA expression in that region differs from all other regions by p

Article Snippet: Quantitative real-time PCR (qPCR) was performed on a CFX Connect Real-Time system (BioRad) using iTag SYBRGreen Supermix (BioRad) by the following primers: col25a1 , 5′- GAT TCT CCT CTT CGG CCT CT -3′and 5′- AAA TAA GAA CGG CCA GGG AG -3′; col23a1 , 5′- GCA ATC AGG ACG AGA TGG CT-3′ and 5′- AAA GTC TCC CGG TGT ACC CT-3′; col17a1 , 5′-TGG GAT CAG CTT TGG GCA TC-3′ and 5′- GAC AAA CCA GCG GCT CGG A-3′; col13a1 , 5′- AAG GGA GAA GCA GGC CTA GAG-3′ and 5′-TGG AGT ACC AGG CAA TCC CAG-3′; gapdh , 5′-CGT CCC GTA GAC AAA ATG GT-3′ and 5′-TTG ATG GCA ACA ATC TCC AC-3′.

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction