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. 2011 Mar 8;39(12):5215–5231. doi: 10.1093/nar/gkr086

Figure 1.

Figure 1.

Rabbit reticulocyte lysate contains RISC components as well as endogenous miRNAs. (A) RISC proteins are present in the RRL. 1, 2 or 3 µl of RRL, HeLa cell S10 and S100 lysates were analyzed by western blotting with antibodies specific for Dicer, Ago2 and PABP as a loading control. TNRC6 was immunoprecipitated and analyzed by western blotting using specific antibodies. An asterisk corresponds to a non-specific band observed for TNRC6 in untreated RRL. (B) Endogenous miRNAs can be detected in untreated RRL. Total RNAs extracted from untreated RRL were hybridized to a microarray containing probes for most known mammalian miRNAs (LC Sciences). Each colored spot corresponds to an miRNA that is present in RRL; a total of more than 300 different miRNAs were detected. Microarray raw data signal is presented for let7, miR-221, miR-155, miR-451 and miR-638 (full data can be found in Supplementary Table S1). (C) Splinted ligation assays against let-7, miR-451 and miR-221 were carried out using increasing amounts of total RNA to validate microarray results. miRNAs not identified by microarray, such as miR-221, were not detected by splinted ligation. (D) Quantification of miRNAs present in untreated RRL. Quantitative PCR was carried out using specific primers against let-7, miR-451 and miR-638.