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. Author manuscript; available in PMC: 2009 Aug 28.
Published in final edited form as: Methods Mol Biol. 2008;419:215–230. doi: 10.1007/978-1-59745-033-1_15

Fig. 2.

Fig. 2

In vitro translation of firefly luciferase mRNA with increasing length poly(A) in HeLa extract. A. Triplicate samples of firefly luciferase mRNA with a non-functional ApppG cap and 0 or 98 nucleotide poly(A) tails (A0, A98), or m7GpppG-capped firefly luciferase mRNA with poly(A) tails of 0, 14, 20, 54 or 98 nucleotides were translated in HeLa cytoplasmic extracts together with a fixed amount of m7GpppG-capped Renilla luciferase mRNA. The relative light units of each luciferase reporter were determined in a luminometer and results with each sample of firefly luciferase were normalized to the internal Renilla luciferase control. The value for m7GpppG-capped firefly luciferase mRNA with no poly(A) (cap A0) was arbitrarily set to one in order to quantify the fold increase in translation observed with increasing length poly(A) on the reporter mRNA. Shown are the mean ± standard deviation for the triplicate determinations. B. Each of the samples in A were pooled and RNA extracted after translation in vitro was analyzed by Northern blot. The blot was hybridized with a mixed probe for firefly (Fluc) and Renilla (Rluc) luciferase mRNA.