Table 7.

Summary of mRNA levels in GFP shRNA control transfectants, URE3-BP shRNA transfectants, and nontransfected HM1:IMSS trophozoites

shRNA transfectant or control sample	URE3-BP 5' oligo pair	P-value	URE3-BP middle oligo pair	P-value	URE3-BP 3' oligo pair	P-value
GFP	100.0 ± 2.9	--	100 ± 2.8	--	100 ± 4.3	--
HM1:IMSS	106.4 ± 5.8	0.2928	108.9 ± 5.6	0.1008	102.8 ± 5.0	0.5792
URE3-BP (350–378)	67.0 ± 2.5	<0.0001	67.4 ± 2.0	<0.0001	72.2 ± 2.8	<0.0001
URE3-BP (580–608)	12.4 ± 0.8	<0.0001	13.5 ± 3.3	<0.0001	12.5 ± 3.8	<0.0001

The average URE3-BP transcript level as measured by qRT-PCR and normalized to actin was defined as being 100% in the GFP shRNA control transfectants. HM1:IMSS nontransfected amebae were also included. Three different oligo pairs amplifying the 5', middle, and 3' sections of URE3-BP were used (sequences and locations are shown in Table 3). Student's t test was used for statistical analysis. Three biological replicates were each assayed in quadruplicate with each oligo pair, with the exception of the HM1:IMSS samples, which had one biological replicate. Values are expressed as the percentage of URE3-BP mRNA of the GFP control shRNA transfectant level ± SE, with the P-value following each.