cdna synthesis  (Thermo Fisher)

Journal: Cancer immunology research

Article Title: Soluble CD80 protein delays tumor growth and promotes tumor infiltrating lymphocytes

doi: 10.1158/2326-6066.CIR-17-0026

Figure Lengend Snippet: CD80-Fc activates transcription factors downstream of CD28 and the TcR. A, CD80-Fc induces transcription of EGR1/2/3/4. CD3 + cells were isolated by negative selection from the blood of healthy human donors, activated with PHA for 72 hours, and then either untreated or incubated for two hours with isotype control mAb, agonist CD28 mAb, TROY-Fc, or CD80-Fc. RNA was then isolated, converted into cDNA, and the DNA analyzed by qRT-PCR for EGR1/2/3/4. Values were normalized to β-actin expression. Results are shown as fold change in expression level compared to untreated T cells. Statistical analysis was performed using Student’s t test. Data are from one of three independent experiments. B, CD3 + CD28+ Jurkat cells were treated for 30 min at 37OC with isotype mAb, agonist CD28 mAb, TROY-Fc, or CD80-Fc. Cells were then fixed and permeabilized, and stained with mAbs to pMAPK or pNF-κB followed by anti-rabbit IgG-FITC. C, Jurkat cells were untreated or treated for 30 min with agonist anti-CD28 mAb and then lysed and analyzed by western blot for phosphorylated MAPK and NFκB. Data for B and C are representative of one of two and one of three independent experiments, respectively.

Article Snippet: RNA was isolated from T cells according to the manufacturer’s protocol and quantified using a BioTek Synergy 2 microplate reader. cDNA was created from each RNA sample using the Maxima cDNA kit (Thermo Scientific) per the manufacturer’s protocol.

Techniques: Isolation, Selection, Incubation, Quantitative RT-PCR, Expressing, Staining, Western Blot

Journal: Scientific Reports

Article Title: Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content

doi: 10.1038/srep05018

Figure Lengend Snippet: Development of AtMYB111 -expressing transgenic tobacco lines and their phenotypic characterization. (a) Schematic representation of T-DNA region of plant expression construct carrying AtMYB111 cDNA in pBI121 vector used for tobacco transformation. (b) Flower color alterations in different transgenic lines. The pigmentation in petals of transgenic lines expressing AtMYB111 (1, 2 and 3) was compared with WT and empty vector (EV) transformed tobacco lines. (c) Confirmation of the presence of the transgene by PCR amplification of transgene using forward and reverse primers of AtMYB111 in different transgenic lines. (d) Expression analysis of transgene through semiquantitative RT-PCR using total RNA from leaves of WT, EV and different transgenic lines. (e) Total anthocyanin content in petals of WT, EV and different transgenic lines. Data are expressed as means ± SE of at least 3 independent experiments, each experiment consisting of 3 technical replicates. In (c) and (d), gels have been cropped for clarity and conciseness of the presentation. *** indicate values that differ significantly from WT at P

Article Snippet: Total RNA was subjected to the reverse transcription reaction to generate first-strand cDNA using oligo(dT) primers (Fermentas Life Sciences, Ontario, Canada).

Techniques: Expressing, Transgenic Assay, Construct, Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line

doi: 10.1074/jbc.M110.107607

Figure Lengend Snippet: CpG methylation status in the 5′ and 3′-LTR regions of BLV proviruses integrated in different infected cell lines. A , mRNA expression levels in the BLV-infected YR2, L267, and L267 LTaxSN cell lines. Total RNA samples were extracted and digested by DNase I. First strand cDNA was synthesized by reverse transcription, and quantitative PCR was performed with oligonucleotide primers amplifying the 5′-UTR region of BLV or the β-actin gene. Results are presented as the ratio of 5′-UTR to β-actin and are the mean values of triplicate samples. The results from a representative experiment of three independent experiments are shown. B , ex vivo expression of BLV in infected YR2, L267, and L267 LTaxSN cell lines. The BLV p24 major gag antigen levels were titrated in the culture supernatants by ELISA. Data are the mean values of triplicate samples. The results from a representative experiment of three independent ELISA experiments are shown. C , bisulfite sequencing analysis of the CpG methylation status in the 5′- and 3′-LTRs of the YR2, L267, and L267 LTaxSN cell lines. Genomic DNA from the three cell lines was extracted and treated by sodium bisulfite/hydroquinone. The two LTRs were amplified by nested PCR, amplified PCR products were subcloned in a TA cloning vector, and 10–11 clones were sequenced for each cell line. The methylation status of these clones is presented for each cell line. The positions and orientations of PCR primers ( NA , NB , NC , ND , NE , NF , NG , and NH ) used to amplify bisulfite-treated BLV DNA by nested PCR are indicated by arrows. Open circles represent unmethylated CpG dinucleotides, and filled circles represent methylated CpG dinucleotides. The −154 CpG and −129 CpG (located within the CRE1 and CRE2 sites, respectively) are hypermethylated in the L267 cell line and are indicated by asterisks .

Article Snippet: First strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen).

Techniques: CpG Methylation Assay, Infection, Expressing, Synthesized, Real-time Polymerase Chain Reaction, Ex Vivo, Enzyme-linked Immunosorbent Assay, Methylation Sequencing, Amplification, Nested PCR, Polymerase Chain Reaction, TA Cloning, Plasmid Preparation, Clone Assay, Methylation

Journal: The Journal of Experimental Biology

Article Title: Muscular tissues of the squid Doryteuthis pealeii express identical myosin heavy chain isoforms: an alternative mechanism for tuning contractile speed

doi: 10.1242/jeb.064055

Figure Lengend Snippet: RNA extraction and cDNA synthesis

Article Snippet: First strand cDNA synthesis was performed using 125–250 ng total RNA with the Moloney murine leukemia virus reverse transcriptase (M-MuLV RT) from the Phusion RT-PCR Kit according to the manufacturer's instructions (Finnzymes, Espoo, Finland).

Techniques: RNA Extraction