Why are you using capillary gel electrophoresis instead of qPCR? The detection limits of capillary tubes is very limited - easy to saturate & minimal signal for detection is also high.

You can use a 1-step qPCR for your splice variants and 1-step for a housekeeping gene.

Dear Katie A S Burnette , Thank you very much for your response. The splicing variants I am studying differ by only a few nucleotides, and the splicing sites are very tricky. I have tried different strategies for primer design, but they are not always specific to each variant. Therefore, I plan to conduct capillary electrophoresis along with qPCR to validate the results using both techniques, due to the lack of primer specificity.

Internal Controls in cDNA Synthesis and Capillary Electrophoresis

Internal controls are essential for normalization to account for variations in RNA quantity, quality, and reverse transcription efficiency. For gene-specific cDNA synthesis during capillary electrophoresis, the following are recommend

1. Housekeeping Genes

Examples: GAPDH, ACTB (beta-actin), or 18S rRNA.

These genes are stably expressed across different conditions and are commonly used as reference genes.

Ensure the housekeeping gene expression is not affected by the DNA-damaging drug treatment.

2. Exogenous Control

Add an external RNA or DNA spike-in control (e.g., luciferase RNA).

This control allows normalization independent of the sample's endogenous variability.

3. Normalization Strategies

Use the delta-delta Ct method if qPCR data is involved to compare expression levels relative to the housekeeping gene.

For capillary electrophoresis, normalize peak areas of the target gene to the internal control.

4. Gene-Specific Validation

Validate the suitability of the chosen housekeeping gene under experimental condition

Understood the lack of primer specificity, and if you are continuing with capillary electrophoresis, please ensure with the target areas of specified gene and try to optimize interms to that of your internal control.