pcr  (Thermo Fisher)

Journal: International Journal of Molecular and Cellular Medicine

Article Title: Overexpression of MiR-138 Inhibits Cell Growth and Induces Caspase-mediated Apoptosis in Acute Promyelocytic Leukemia Cell Line

doi: 10.22088/IJMCM.BUMS.7.1.24

Figure Lengend Snippet: miR-138 up regulation leads to hTERT suppression in NB4 cells. A: total RNAs from NB4 cells that were transduced with GFP hsa-miR-138-expressing lentiviruses were extracted at 96 h after removal of the lentivirus-containing medium. hTERT mRNA expression was measured by qRT-PCR. Values were normalized to GAPDH . (n=3; *P

Article Snippet: The prepared cDNA was subjected to quan-titative reverse-transcriptase polymerase chain reaction (qRT-PCR), using Maxima SYBR Green Master mix (Thermo Scientific, Waltham, Massa-chusetts, USA) in the Rotor Gene 6000 Real Time PCR inst rument (Corbett Research, Hilden, Germany).

Techniques: Transduction, Expressing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

doi: 10.1038/s41467-018-06404-w

Figure Lengend Snippet: MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD ( n = 3). P -value

Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

doi: 10.1038/s41467-018-06404-w

Figure Lengend Snippet: Tethered MARF1 shortens reporter mRNA poly-A tail and reduces reporter protein level. a GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, GFP-coding sequence, and a 3′ UTR containing five BoxB hairpins. LambdaN-HA-fused control peptide, MARF1, GW182, and Piwi under a UASP promoter were expressed in germline cells using maternal-alpha-tubulin-Gal4 driver. b Confocal images of GFP signal in stage 14 oocytes. Scale bar is 100 μm. c Western blots using stage 14 oocyte lysates. Black triangles indicate the transgenic lambda-HA-fused proteins. d Quantification of band intensities in c . e ePAT assay measuring GFP reporter mRNA poly-A tail length in stage 14 oocytes. The amplified DNA sizes were analyzed on an agarose gel. f ePAT assay measuring poly-A tail lengths of GFP reporter mRNA and a negative control cdk1 mRNA in stage 14 oocytes. Amplified DNA sizes were analyzed by capillary fragment analysis. g Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR. Mean ± SD ( n = 6 for the control and n = 4 for all the others)

Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

Techniques: Sequencing, Western Blot, Transgenic Assay, Amplification, Agarose Gel Electrophoresis, Negative Control, Quantitative RT-PCR

Journal: OncoTargets and therapy

Article Title: Nuclear factor-κB p65 regulates glutaminase 1 expression in human hepatocellular carcinoma

doi: 10.2147/OTT.S167408

Figure Lengend Snippet: Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

Article Snippet: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Upon relative treatment, cells were lysed, and total RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Characterization of a Species-Specific Insulinase-Like Protease in Cryptosporidium parvum

doi: 10.3389/fmicb.2019.00354

Figure Lengend Snippet: Production, purification, and characterization of the INS19 fragment expressed by cgd6_5510 . (A) Electrophoresis analysis of a PCR product from the cgd6_5510 region. Lane M: DNA marker; Lane 1: cgd6_5510 PCR product. (B) The Western blot analysis of the expression of recombinant INS19 in Escherichia coli Rosetta (DE3). Lane M: protein marker; Lane 1: lysate of un-induced bacterial cells transformed with pET28s- cgd6_5510 ; Lane 2: supernatant from lysate of bacterial cells induced by IPTG; Lane 3: inclusion bodies from lysate of bacterial cells induced by IPTG. (C) Purification of recombinant INS19 in inclusion bodies as indicated by results of SDS-PAGE analysis. Lane M: protein marker; Lane 1: proteins from inclusion bodies dissolved in wash buffer containing 1 M urea; Lane 2: proteins from inclusion bodies dissolved in wash buffer containing 2 M urea; Lane 3: proteins from inclusion bodies dissolved in buffer containing 8 M urea; Lane 4: undissolved proteins from inclusion bodies after washing; Lane 5: purified recombinant INS19 after dialysis and ultrafiltration. (D) The Western blot analysis of purified INS19 (Lane 1), C. parvum sporozoite lysate (Lane 2), and C. parvum oocyst lysate (Lane 3) probed with the pre-immune serum (left panel) or polyclonal anti-INS19 antiserum (right panel). Lane M: protein marker.

Article Snippet: The PCR analysis of DNA and cDNA was performed in duplicates with the Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) under the following conditions: denaturation at 95°C for 5 min; 35 cycles of amplifications at 95°C for 45 s, 50°C for 45 s, and 72°C for 60 s; and a final extension at 72°C for 7 min.

Techniques: Purification, Electrophoresis, Polymerase Chain Reaction, Marker, Western Blot, Expressing, Recombinant, Transformation Assay, SDS Page