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.

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

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