Expression Analysis

Western Blotting

Horizon will perform western blotting on one protein from one standard tissue from both your SAGE® research model and wild type control with n=3. Three technical replicates are performed, and three concentrations of primary antibody are examined. You provide the primary antibody; however we can obtain the primary antibody for you for an additional fee. Actin will be used as a loading control.

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You receive1

  • Digital gel image with actin control
  • Complete protocol, including tissue harvest, protein extraction, antibody concentrations, electrophoresis and transfer conditions, exposure times, etc.
 

Figure 1. Western blotting was performed in MeCP2 knockout rats (lane 2) and wild type controls (lane 1).

 

qRT-PCR

Probe-based qRT-PCR on one gene from one standard tissue from both your SAGE research model and wild type control with n=3. Three technical replicates are performed, and two primer/probe sets will be used per analysis targeting discrete regions of the transcript. qRT-PCR on the housekeeping gene cyclophilin will also be performed.

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You receive2

  • Raw data including standard curve
  • Graph of SAGE research model vs. control
  • Protocol, including tissue harvest, RNA extraction, isolation, and QC, primer/probe sequence or product number, and cycling conditions

Figure 2. qRT-PCR was performed in Pink1 knockout rats and wild type controls.

1 For western blotting, the gel image is guaranteed to show a clear band at the expected molecular weight in positive control. One primary antibody will be used at a maximum of three different concentrations. If no clear band is seen at the expected MW after three concentrations of primary antibody, the project will be terminated. Additional antibodies can be tried at an additional cost of $1900/antibody. No guarantees are made on experimental results—e.g. non-specific bands may be seen in knockout samples. 

2 For qRT-PCR, intra-assay variability is guaranteed at <10% for technical replicates with R2 >0.97. No guarantees are made regarding experimental results, including the amount of transcript present in your SAGE model.

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