Novel RNA interference methods using dna-RNA duplex constructs
a duplex construct and rna technology, applied in the field of new rna interference methods using dna-rna duplex constructs, can solve the problems of single-stranded dna antisense oligonucleotides with relatively short-term effectiveness, inability to achieve the effect of biological effect, so as to reduce the occurrence of interferon-induced cytotoxicity and alter the genetic characteristic of eukaryoti
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example 1
Cell Fixation and Permeabilization
[0114]LNCaP cells, a prostate cancer cell line, were grown in RPMI 1640 medium supplemented with 2% fetal calf serum. A sample containing cells cultured in a 60 mm dish (70% full of cells) was trypsinized, collected and washed three times in 5 ml phosphate buffered saline (PBS, pH 7.2) at room temperature. After washing, the cells were suspended in 1 ml of ice-cold 10% formaldehyde solution in 0.15M NaCl. After one-hour incubation on ice with occasional agitation, the cells were centrifuged at 13,000 rpm for 2 min, and washed three times in ice-cold PBS with vigorous pipetting. The collected cells were resuspended in 0.5% non-ionic detergents, such as (octylphenoxy)-polyethanol or polyoyethylenesorbitan (Sigma), and incubated for one hour with frequent agitation. The cells were washed three times in ice-cold PBS containing 0.1M glycine, then resuspended in 1 ml of the same buffer with vigorous pipetting in order to be evenly separated into small ali...
example 2
In-Cell Reverse Transcription and Poly-(N) Tailing of cDNAs
[0115]For reverse transcription of mRNAs in cells, twenty of the fixed cells were thawed, resuspended in 20 μl of ddH2O, heated to 65° C. for 3 min and then cooled on ice. A 50 μl RT reaction was prepared, comprising 5 μl of 10× in-cell RT buffer (1.2M KCl, 0.5M Tris-HCl, 80 mM MgCl2, 10 mM dithiothreitol, pH 8.1 at 42° C.), 5 μl of 5 mM dNTPs, 25 pmol oligo(dT)n-T7 promoter (SEQ ID NO. 1), 80 U RNase inhibitor and above cold cells. After reverse transcriptase (40U) was added, the RT reaction was mixed and incubated at 55° C. for three hours. The cells were then washed once with PBS and resuspended in a 50 μl tailing reaction, comprising 2 mM dGTP, 10 μl of 5× tailing buffer (250 mM KCl, 50 mM Tris-HCl, 7.5 mM MgCl2, pH 8.3 at 20° C.). The tailing reaction was heated at 94° C. for 3 min and then chilled in ice for mixing with terminal transferase (20 U), following further incubation at 37° C. for 20 min. Final reaction was s...
example 3
Single-Cell mRNA Amplification
[0116]To increase the intracellular copies of whole mRNAs, the T7 promoter region of a poly(N)-tailed cDNA was served as a coding strand for the amplification by T7 RNA polymerase (Eberwine et al., Proc. Natl. Acad. Sci. USA 89: 3010-3014 (1992)). As few as one cell in 5 μl of the above tailing reaction can be used to accomplish full-length aRNA amplification. An in-cell transcription reaction was prepared on ice, containing 25 pmol poly(dC)-12mer primer (SEQ ID NO. 2), 1 mM dNTPs, Pwo DNA polymerase (5U), 5 μl of 10× Transcription buffer (Roche), 2 mM rNTPs and T7 RNA polymerase (2000U). The hybridization of 20mer primer to the poly(N)-tailed cDNAs was incubated at 65° C. for 5 min to complete second strand cDNA synthesis and then RNA polymerase was added to start transcription. After four-hour incubation at 37° C., the cDNA transcripts were isolated from both cells and supernatant, to be directly used in the following reverse transcription. The reacti...
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