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Cleavage and polyadenylation of the β-globin transcripts.

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posted on 12.07.2010, 00:23 authored by Andrea B. Eberle, Viktoria Hessle, Roger Helbig, Widad Dantoft, Niclas Gimber, Neus Visa

(A) Detection of the downstream cleavage product (3′ fragment). The expression of Rat1 was silenced by RNAi in S2 cells expressing either mut or wt β-globin. Control cells were treated in parallel with GFP-dsRNA. Total RNA was purified and reverse-transcribed from dsRNA-treated cells, and the resulting cDNAs were quantified by qPCR with primers specific for the pre-mRNA, the mRNA (see Figure 4A) and the 3′ fragment (see the schematic picture above the histogram). The histogram shows the average values and standard deviations of β-globin levels, normalized to actin 5C RNA, from three independent experiments with two qPCR runs each (n = 6). The relative accumulation of downstream product was less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01 using a paired, two-tailed Student's t-test, n = 6). (B) PCR-based detection of polyadenylated β-globin transcripts. Total RNA was purified from nuclear preparations of S2 cells expressing either mut or wt β-globin genes. The resulting cDNAs were used to amplify polyadenylated β-globin sequences by PCR with a 26 nt-long downstream primer complementary to the beginning of the poly(A) tail, as indicated in the figure (lanes 14). Control reactions with a 16 nt-long primer lacking the oligo(dT) extension were processed in parallel to rule out poly(A)-independent priming (lanes 58). Contamination with genomic DNA was assessed in parallel reactions without reverse transcriptase (RT-). (C) RT-qPCR quantification of polyadenylated β-globin transcripts. The samples described above were analyzed by RT-qPCR. Relative levels of polyadenylated β-globin transcripts, normalized to the total β-globin RNA, are shown. Values and standard deviations represent the average from two independent experiments with two qPCR runs each (n = 4). No significant differences were observed using a paired, two-tailed Student's t-test (p = 0.12, n = 4).