steponeplus real time polymerase chain reaction system  (Thermo Fisher)

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: IGSF3 regulation of sphingolipid metabolism. ( A and B ) Heatmap of expression levels of genes involved in sphingolipid ( A ) and ganglioside ( B ) metabolism relative to endogenous IGSF3 expression levels, measured by cDNA microarray in lymphoblastoids from patient and 5 healthy controls. ( C–G ) mRNA expression levels of indicated enzymes determined by targeted qPCR. Dots represent independent experiments; mean ± SEM shown as horizontal lines; * P

Article Snippet: A total of 2 μg of total RNA (RNeasy Mini Plus, QIAGEN) was used to reverse transcribe cDNA (High-Capacity cDNA Reverse Transcription, Thermo Fisher Scientific). qPCR was then performed on the cDNA (StepOnePlus System, Thermo Fisher Scientific) using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) and probes (all from Thermo Fisher Scientific) specific for human IGSF3 (Hs00155437_m1, lot no. 1163927), mouse IGSF3 (Mm01302155_m1, lot no. 1342279), human CERT (Hs01062552_m1, lot no. 156453), human FAPP2 (Hs01696164_s1, lot no. P161121-011 A12), human ITGB1 (Hs00559595_m1), and human GBA (Hs00164683_m1, lot no. 1611503).

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

Journal: Molecular Medicine Reports

Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

doi: 10.3892/mmr.2018.9141

Figure Lengend Snippet: Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time PCR and TaqMan ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.

Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

Techniques: Genotyping Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Fluorescence, Software, Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

doi: 10.3892/mmr.2018.9141

Figure Lengend Snippet: Detection of the BRCA1 ex9-12del mutation by real-time PCR and TaqMan ® probe. Red amplification curves represent the fluorescent signal given by the FAM ™ dye from the TaqMan ® probe detecting the variant allele in the four positive samples, which had a mean Ct of 27.46. No amplification curve is observed for the negative samples neither for negative controls (horizontal red lines). The ‘Y’ axis values represent the fluorescent signal normalized of the FAM ™ dye with the ROX, whereas the ‘X’ axis represent number of cycles. PCR. PCR, polymerase chain reaction; ROX, passive reference dye.

Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Amplification, Variant Assay, Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo

doi: 10.4049/jimmunol.1502399

Figure Lengend Snippet: p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

Article Snippet: QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System (Thermo fisher) using pre-designed TaqMan® Gene Expression Assays (Life Technologies) (18S RNA Mm03928990 and p21 Mm04205640) according to manufacturer's instructions.

Techniques: Isolation, Cell Culture, Recombinant, FACS, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing