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Table 5 Summary of Igl mRNA levels in Igl shRNA transfectants

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

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
  1. 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.