Synthesis of modified siRNA bearing C-5 polyamine-substituted pyrimidine nucleoside in their 3′-overhang regions and its RNAi activity

Bioorganic and Medicinal Chemistry Letters

Volumn 21, Issue 2, 2011, Pages 715-717

  Masud, Mohammad Mehedi ^a,b   Masuda, Tomokazu ^a   Inoue, Yusuke ^a   Kuwahara, Masayasu ^a   Sawai, Hiroaki ^a   Ozaki, Hiroaki ^a  

^a GUNMA UNIVERSITY   (Japan)

^b UNIVERSITY OF DHAKA   (Bangladesh)

Author keywords

HNF4 ; Modified CPG support; Modified siRNA; RNAi; siRNA

Indexed keywords

5 BIS(AMINOETHYL)AMINOETHYLCARBAMOYLMETHYL 2' DEOXYURIDINE; HEPATOCYTE NUCLEAR FACTOR 4ALPHA; NUCLEOSIDE; SMALL INTERFERING RNA; THYMIDINE; UNCLASSIFIED DRUG; URIDINE DERIVATIVE;

ARTICLE; DRUG SYNTHESIS; GENE EXPRESSION; GENE SILENCING; HUMAN; HUMAN CELL; RNA INTERFERENCE; SUBSTITUTION REACTION;

HEP G2 CELLS; HEPATOCYTE NUCLEAR FACTOR 4; HUMANS; POLYAMINES; PYRIMIDINE NUCLEOSIDES; RNA INTERFERENCE; RNA, MESSENGER; RNA, SMALL INTERFERING;

EID: 78651225247     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.11.125     Document Type: Article

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12. RNAi activity: HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% FBS at 37 °C. Each siRNA was then transfected into the cells with transfection reagent (Hily Max, DOJINDO). After incubation at 37 °C for 24 h, medium was changed to DMEM containing 10% FBS. After additional 24 h incubation, total RNA was extracted from the cells using RNAiso Plus (TaKaRa). The RNA was reverse-transcribed using ReverTraAce qPCR RT kit (TOYOBO). Real-time PCR was performed on a MyiQ Real-time PCR detection system (Bio-Rad). Purified cDNAs encoding HNF4α and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified using THUNDERBIRD SYBR qPCR Mix (TOYOBO) with specific primers (HNF4α: 5′-CAGGCTCAAGAAATGCTTCC-3′ and 5′-GGCTGCTGTCCTCATAGCTT-3′; GAPDH: 5′-CAATGACCCCTTCATTGACC- 3′ and 5′-GACAAGCTTCCCGTTCTCAG-3′). PCR was performed at 95 °C for 1 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. For each sample, the mean threshold cycle (Ct) from two replicate PCR using RNA isolated from independent cells was taken. Expression levels of HNF4α mRNA were normalized to GAPDH by the ΔΔCt method.