Kindly confirm how can we interpret data of delta Ct in RT-PCR Results?
We have just control tissue, not any test sample; thereby delta delta Ct values can't be find out.
We hv just delta Ct values (house keeping and test gene of control tissue only).
How to interpret the data with delta Ct values only ?
Delta Ct is the difference between the Ct values of a target gene and a reference gene (also called a housekeeping gene) in the same sample. Delta Ct values can be used to compare the relative expression levels of a target gene across different samples. The lower the delta Ct value, the higher the expression of the target gene relative to the reference gene. However, delta Ct values do not provide information about the absolute expression levels of a target gene or the fold change in expression between different samples. For that, you need to use delta-delta Ct values, which require a control sample and a test sample. If you only have control samples, you can only compare the relative expression of the target gene within those samples using delta Ct values.
Some factors that can affect the interpretation of delta Ct values are:
- The choice and quality of the reference gene. The reference gene should be stably expressed across different samples and not affected by experimental conditions. It should also have similar amplification efficiency as the target gene.
- The quality and quantity of the input RNA. The input RNA should be free of contaminants, such as genomic DNA or inhibitors, that can affect the RT-PCR reaction. The amount of input RNA should be normalized across different samples to avoid bias in delta Ct values.
- The amplification efficiency and specificity of the RT-PCR reaction. The amplification efficiency should be close to 100% for both target and reference genes, and the specificity should be verified by melting curve analysis or gel electrophoresis. The Ct values should be within the linear range of the standard curve.
To improve the accuracy and reliability of delta Ct values, it is recommended to use multiple reference genes and multiple biological replicates for each sample.