Confirmation that the CIRP long 5′-UTR does not result in aberrant splicing events or have promoter activity. (A) RT-PCR using a primer upstream of the pRF intron shows only the bicistronic mRNA is present and no aberrant splicing products are detected from the long 5′-UTR transfected cells or pRF controls. (B) Cloning of the long 5′-UTR into a promoterless vector results in a very small amount of activity that does not account for the increase in expression of the downstream cistron. (C) Northern analysis using a firefly specific probe shows identical profiles for control (pRF, pRMF) and test (pRF-L, pRF-S) samples. (D) Loading control for Northern blots shown in C. (E) The ratio of firefly to renilla mRNA transcript level as determined by qRT-PCR in the presence of the short (pRF-S) or long (pRF-L) CIRP 5′-UTR in the intercistronic region of the pRF expression vector. (F) A normal PCR confirming that a dicistronic message of the correct size is generated from pRF-L and that full-length renilla and firefly open reading frames are produced. For further details see the text.
