Journal: Immunogenetics

Article Title: Identification and characterization of a FasL-like protein and cDNAs encoding the channel catfish death-inducing signaling complex

doi: 10.1007/s00251-004-0701-2

Figure Lengend Snippet: Expression of FasR, FADD, and caspase 8 in PBL and various catfish LCL. Total RNA was isolated from T (G14D), cytotoxic T (TS 32.17), B (1G8, 3B11), macrophage (42TA), and fibroblast (CCO) cell lines as well as PBLs from two fish and reverse transcribed using an oligo[dT] primer. The resulting cDNA was used in PCR along with gene-specific primers. Actin was included as a positive control. The presence or absence of FasL cross-reactive proteins as determined previously by Western blotting is indicated: + 37 denotes the 37,000- M r membrane form of FasL, whereas + 15 denotes the 15,000- M r soluble form. ND denotes not detected

Article Snippet: Total RNA (2.5 μg) was reverse transcribed with an oligo(dT) primer and Superscript II RNase H-minus reverse transcriptase (Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Isolation, Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Positive Control, Western Blot

Journal: BMC Genomics

Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

doi: 10.1186/1471-2164-9-94

Figure Lengend Snippet: Spearman's rank correlation tree of the gene expression patterns from amplified and unamplified RNA. RNA was amplified from 500, 50 and 5 ng total RNA using the oligo-dT and ADP methods. All M. tuberculosis H37Rv genes were clustered using median expression ratios from 2–6 replicate hybridisations.

Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

Techniques: Expressing, Amplification

Journal: BMC Genomics

Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

doi: 10.1186/1471-2164-9-94

Figure Lengend Snippet: The size distribution of products amplified from 500 (triangle), 50 (square), 5 ng (diamond) M. tuberculosis total RNA using oligo-dT (A) and ADP (B) amplification strategies. (C) Unamplified total mycobacterial RNA. The peaks at 25 bp represent the marker added to all samples. The size distributions were plotted using the average of 2–4 replicate amplifications. Abundance units are detailed in relative fluorescence and plotted against migration time that has been converted into a base pair (bp) estimate of product size as measured using the Agilent Bioanalyser.

Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

Techniques: Amplification, Marker, Fluorescence, Migration

Journal: BMC Genomics

Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

doi: 10.1186/1471-2164-9-94

Figure Lengend Snippet: A comparison of the number of genes not detected by microarray analysis from unamplified RNA or from products generated by oligo-dT and ADP amplification methods using 500 or 50 ng starting total RNA. The number of genes below a 2 fold signal/background threshold in the RNA channel was compared between amplified and unamplified gene expression signatures as a measure of the potential transcriptome data lost during amplification.

Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

Techniques: Microarray, Generated, Amplification, Expressing

Journal: BMC Genomics

Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

doi: 10.1186/1471-2164-9-94

Figure Lengend Snippet: A Spearman's rank correlation of the 155 genes identified to be significantly differentially expressed in microaerophilic compared to aerobic M. tuberculosis growth conditions using unamplified RNA (marked Un). The mean gene expression ratios derived from unamplified RNA and from the products of 500, 50, and 5 ng amplifications using oligo-dT and ADP methods are displayed. Genes are ordered in rows, amplification conditions as columns. Red colouring indicates genes induced in microaerophilic vs. aerobic growth conditions; green colouring denotes repression.

Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

Techniques: Expressing, Derivative Assay, Amplification

Journal: The Journal of general virology

Article Title: Ovine herpesvirus-2 encoded microRNAs target virus genes involved in virus latency

doi: 10.1099/vir.0.059303-0

Figure Lengend Snippet: A. Genomic location of ORF 20 and ORF 50 relative to adjacent genes. Genes are indicated by open boxes, arrow head represents direction of transcription. Dotted lines represent introns. The nucleotide positions representing the location of the predicted TATA box and poly A site for each gene are indicated. The position of primers used for PCR and cDNA priming (ORF 20) are indicated by arrows and nucleotide position. Predicted miRNA binding sequences are indicated by vertical bars in the respective UTRs. B. ORF 20 Lane 1, No RT; Lane 2, cDNA primed with 35 pm primer (250 ng); Lane 3, cDNA primed with 10 pm primer (66ng); Lane 4,: No template control; Lane 5, DNA positive control. Lane 6; Marker, Generuler 100bp C. ORF 50 Lane 1, No RT, Lane 2, cDNA primed with Oligo dT; Lane 3, cDNA primed with random primers; Lane 4, No template control; Lane 5, DNA positive control; Lane 6, Marker, Generuler 1kb D. ORF73 Lane 1, No RT, Lane 2, cDNA primed with Oligo dT; Lane 3, cDNA primed with random primers; Lane 4, No template control; Lane 5, DNA positive control; Lane 6, Marker, Generuler 100bp

Article Snippet: 1 μg of RNA was digested with RQ1 Dnase (Promega) for 30 min at 37 °C. cDNA was primed using 250ng Oligo dT primer (Promega; equivalent to 0.5 μg/μg RNA) and synthesized using AMV reverse transcriptase for 1 hr at 42 °C.

Techniques: Polymerase Chain Reaction, Binding Assay, Positive Control, Marker

Journal: Nucleic Acids Research

Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

doi: 10.1093/nar/gkq131

Figure Lengend Snippet: Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

Article Snippet: RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany).

Techniques: Sequencing, Migration, Modification, Hybridization

Journal: BMC Genomics

Article Title: Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

doi: 10.1186/1471-2164-12-196

Figure Lengend Snippet: KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

Article Snippet: RT-PCR and qRT-PCR Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided.

Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing, Marker, Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene

doi: 10.1371/journal.pone.0022727

Figure Lengend Snippet: The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

Article Snippet: Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions.

Techniques: Plasmid Preparation, Luciferase, Expressing, Transfection, Quantitation Assay, Quantitative RT-PCR, Western Blot

Journal: The Journal of Cell Biology

Article Title: Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component

doi: 10.1083/jcb.200211083

Figure Lengend Snippet: p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. cDNA generated from oligo-dT-primed total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.

Article Snippet: For RT-PCR, RNA was isolated from 20 larvae using TRIzol reagent and the manufacturer's protocol (Invitrogen Life Technologies), cDNA was generated using oligo-dT primers (Promega Reverse Transcription System), and 5 of 100 μl used for PCR.

Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Generated