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.

Linford et al. BMC Microbiology 2009 9:38   doi:10.1186/1471-2180-9-38