Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

BMC Microbiology

Table 5 Summary of Igl mRNA levels in Igl shRNA transfectants

shRNA transfectant or control sample	Igl 5' oligo pair	P-value	Igl 3' oligo pair	P-value	Igl1 oligo pair	P-value	Igl2 oligo pair	P-value
GFP6	100.0 ± 4.1	--	100.0 ± 4.9	--	100.0 ± 3.0	--	100.0 ± 4.0	--
HM1:IMSS	101.4 ± 4.3	0.7741	96.1 ± 3.5	0.3239	105.5 ± 3.1	0.1382	103.9 ± 6.1	0.5713
Igl (2412–2440)	100.6 ± 5.0	0.9172	103.4 ± 9.1	0.7717	91.1 ± 6.9	0.2426	106.0 ± 5.2	0.2919
Igl1 (272–300)	71.3 ± 2.9	<0.0001	67.1 ± 3.0	<0.0001	61.1 ± 3.2	<0.0001	70.2 ± 2.7	<0.0001
Igl (1198–1226)	70.9 ± 2.7	<0.0001	62.1 ± 1.6	<0.0001	68.3 ± 2.5	<0.0001	76.8 ± 1.6	<0.0001
Igl (2777–2805)	68.1 ± 3.3	<0.0001	62.3 ± 2.9	<0.0001	74.1 ± 3.3	<0.0001	77.8 ± 3.0	<0.0001

For qRT-PCR, samples were amplified with the actin oligo pair as a control, or with four pairs of Igl oligos: Igl 5', amplifying the 5' end of both Igl1 and Igl2, Igl 3', amplifying both Igl1 and Igl2 at the 3' end, and oligos specific for Igl1 and Igl2 individually, amplifying Igl1- or Igl2-specific sequences near the 5' end. Oligo sequences are shown in Table 3. Three biological replicates were each assayed in quadruplicate sets with each oligo pair, with the exception of the HM1:IMSS samples, which had one biological replicate. Igl and actin levels were calculated by using both the relative standard curve and the ΔΔC(t) method [54, 55] and actin was used as the normalization control. The average level of Igl in the GFP control shRNA transfectants was defined as 100% expression of Igl mRNA for computational purposes. Igl levels in the Igl transfectant samples and nontransfected HM1:IMSS were compared to the GFP control, and are shown as the percentage of Igl mRNA relative to the GFP control (± SE). Statistical analysis was performed using Student's t test (two-tailed), groups were compared using ANOVA, and the GraphPad QuickCalcs P-value calculator [53] was used to calculate P-values.

ISSN: 1471-2180