Authors
Y.C. Bor, J. Swartz, Y. Li, J. Coyle, D. Rekosh, and Marie-Louise Hammarskjold
Summary
Here we describe a protocol that can be used to determine whether specific mammalian mRNAs are associated with polyribosomes. In the referenced Nature paper, this method was used to show that Tap/NXF1 mRNA retaining intron 10 is present in polyribosomes. This method can also be used to determine if mRNAs are differentially associated with the translation machinery under varying conditions. A similar protocol was previously utilized in our laboratory to show that Tap/NXT1 and WT1(+KTS) proteins promote translation of CTE-containing RNA with retained introns (Jin et al., 2003, Bor et al., 2006). The protocol consists of a method for the generation of cytoplasmic extracts from mammalian cells (in this case, 293T cells) without the disruption of polyribosomes, the separation of ribosomal components and polyribosomes by sucrose gradient centrifugation, the isolation of mRNA from these fractions, and detection of mRNA by Northern blot analysis (or alternatively, Reverse Transcriptase-Polymerase Chain Reaction, RT-PCR).Further details
The protocol was published on Protocol Exchange in 2006. To see the entire protocol, click on the source link.Advertisement
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