Archives

  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br Hybridization of oligonucleotides br To study the use of

    2020-08-12


    4.4. Hybridization of oligonucleotides
    To study the use of dUHBI-labeled oligonucleotide ON1 as hybridization-probe, we used custom made complementary 20-OMe-RNA. Solutions of labeled single-strands were mixed at room temperature with an equimolar amount of the complementary single strand oligonucleotide in PBS buffer (pH 7.4). Samples were hybridized by heating to 90 C for 5 min and subsequently allowed to cool to room temperature over 2 h prior to measurements.
    4.5. Absorption and fluorescence measurements
    Absorption spectra of the probes were measured in PBS buffer containing 8.0 g NaC1, 0.2 g KC1, 1.15 g Na2HPO4, 0.2 g KH2PO4, in water 100 mL (pH 7.4). The concentration of the oligonucleotide was 2 mM. Absorbance was kept less than 0.05 AU in order to avoid inner filter distortion. Samples were measured in a 10 mm quartz cell. The fluorescence measurement conditions of oligonucleotide included 710 V sensitivity and a 5 nm slit. Samples were measured in PBS buffer (pH 7.4). lex 390 nm, lem 486 nm. The concentration of the samples was in the range of 0.2 mM. Samples were measured in a 10 mm quartz cell. The quantum yields (F) of the single-stranded and double-stranded oligonucleotides were calculated from the observed absorbance and the area of the fluorescence emission band. The fluorescence quantum yields of all oligonucleotide were determined relative to fluorescein of quinine sulfate in 0.05 M H2SO 4 (labs 390 nm, lem 480 nm, F 0.54). The quantum yield was calculated according to the following equation:
    Here, F and FR are the fluorescence quantum yield of the sample and the reference, respectively, I and IR are areas under the fluorescence spectra of the sample and of the reference, respec-tively, OD and ODR are the Necrostatin1 values of the sample and the reference at the excitation wavelength, and h and hR are the refractive index for the respective solvents used for the sample and the reference. Fluorescence measurements of the probe with the complementary mRNA in RNA extracts were performed at room temperature in PBS buffer (pH 7.4). Total RNA extracts were ob-tained using Tri-Reagent (Sigma) and DNA was removed using Turbo-Dnase Free Kit (Invitrogen). The final concentration of the probe was 0.2 mM. The fluorescence was measured upon excitation at 390 nm. Samples were measured in a 10 mm quartz cell with a 1 cm path length. Duplicate samples were measured and the ex-periments were performed on three separate days.
    Human U2OS cells were maintained in low glucose Dulbecco's modified Eagle's medium (DMEM, Biological Industries, Israel) containing 10% fetal bovine serum (FBS, HyClone), and 100 IU/mL Penicillin, and 100 mg/mL Streptomycin (Biological Industries). Human CAMA-1 cells were maintained in Eagle's Minimum Essential Medium (EMEM, Biological Industries, Israel) containing 10% fetal bovine serum, and 100 IU/mL Penicillin, and 100 mg/mL Streptomycin. Human ZR-75-30 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 Medium (RPMI 1640, Bio-logical Industries, Israel) containing 10% fetal bovine serum, and 100 IU/mL Penicillin, and 100 mg/mL Streptomycin.
    Acknowledgment
    This Research was supported by the Israeli Ministry of Science and Technology. 
    Appendix A. Supplementary data
    References
    Contents lists available at ScienceDirect
    Biomedicine & Pharmacotherapy
    journal homepage: www.elsevier.com/locate/biopha
    An oncogenic gene, SNRPA1, regulates PIK3R1, VEGFC, MKI67, CDK1 and T other genes in colorectal cancer
    Qingmin Zenga, Fuming Leib, Yigang Changa, Zhaoya Gaob, Yanzhao Wangb, Qingkun Gaob, Pengfei Niub, Qiang Lia,
    a National Clinical Research Center for Cancer & Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China b Department of General Surgery, Gastrointestinal Surgery, Peking University Shougang Hospital, Jin Yuan Zhuang Road No. 9, Beijing 100144, China
    Keywords:
    Colorectal cancer
    SNRPA1
    RKO
    Microarray analysis 
    Purpose: Colorectal cancer (CRC) caused more than 65,000 mortalities worldwide per year. It is a result of one or a combination of chromosomal instability, CpG island methylator phenotype, and microsatellite instability. SNRPA1 (small nuclear ribonucleoprotein polypeptide A) is a subunit of spliceosome complex that is involved in the RNA processing. Overexpression of SNRPA1 has been implicated in a variety of cancers including CRC. Besides from its role in mediating the RNA processing, the other aspects regarding its function in the progression of colorectal cancer have not been revealed.
    Methods: Herein, we combined regular gene overexpression or knock down in vitro and in vivo and microarray gene profiling analysis to decipher the unknow regulatory role of SNRPA1 in CRC.