Hello Mansi 

as explained by me to another person with an identical question referenced on this forum below Ct values will or can vary by 1-2 cycles between different samples because of differences in quality and quantity/ copy number of RNA between cDNA samples due to differences in the quality of starting RNA and thus the efficiency of the RT reaction affecting the quality of your final cDNA sample

1.  As a rule therefore different cDNA samples can vary by 1-2 cycles between samples. 

2.  In contrast Ct values for ( say ) a housekeeping gene should always be within 0.5 cycles if derived from the same cDNA sample

3.  Moreover whatever the exact Ct value it must remain constant for the same samples within and between real time assays

4.  Finally to ensure accurate normalisation and gene expression given that different cDNA samples can vary slightly in Ct value ( due to quality differences as explained) on every real time assay GOI amplification data should always be normalised with housekeeping data derived from the same cDNA sample. In other words for different cDNA samples - treated untreated or just different biological replicates of both - in every assay you must amplify your gene of interest and your housekeeping gene from the same cDNA sample to ensure accurate normalisation for each data set in the assay 

https://www.researchgate.net/post/Any_advice_on_the_difference_in_the_Ct_value_of_reference_gene_in_treated_and_control_samples

Hi,

There might be some variation in the Ct values of the housekeeping gene..even if it is not affected by the treatment. The reason cane be many  including the quality of RNA and cDNA preparation. Some variation can be taken care of by normalizing the expression of the test gene with the reference gene in each sample. However, if the housekeeping gene is substantially affected..it is better not to use it for analysis. It is advisable to use a gene which is not affected by the particular treatment. Additionally, it is now a days very common to use more than one reference gene for such analysis..

bye

Hi Mansi,

Absolutely, your housekeeping gene is going to be used to normalized the data and can not be affected by the treatment you are using. Changes in expression of the housekeeping gene will affect the final readout of your gene of interest. In general GAPDH is a particularly bad housekeeping gene in many situations, for example when comparing tissues with different metabolic demands or if a treatment has any effect on basal metabolism. There are many good papers and reviews covering this topic in which you can find a list of housekeeping genes and which one is best for each situation (comparing same tissue sample for two different animals, different tissue samples, different treatments, etc.). I would highly recommend you to find a recent review and decide from there what would be your best option; in general using rRNA is a common practice. Also, as Ajay mentions, nowadays to publish in a high impact journal you might need to use 2 o 3 housekeeping genes simultaneously to verify that your treatment over the housekeeping gene is not compromising your result.

Hope this helps,

Ezequiel 

Hi Mansi,

Like Ezequiel said, the housekeeping gene should not be affected by your treatments. Otherwise, it's not the housekeeping gene for your case.

First, make sure your RNA quality is good. Any contamination or degradation will ruin your result.

Second, make sure your RNA quantification is accurate. You have to put exactly the same amount of RNA of cDNA transcribing.

Last, prepare your RT-PCR reaction mix in parallel. Add exactly the same amount of cDNA into your samples.

After all these, if you still get Ct values variations, GAPDH might not be a good reference for your case. You may have to try other reference genes.

Hello Mansi 

as explained by me to another person with an identical question referenced on this forum below Ct values will or can vary by 1-2 cycles between different samples because of differences in quality and quantity/ copy number of RNA between cDNA samples due to differences in the quality of starting RNA and thus the efficiency of the RT reaction affecting the quality of your final cDNA sample

1.  As a rule therefore different cDNA samples can vary by 1-2 cycles between samples. 

2.  In contrast Ct values for ( say ) a housekeeping gene should always be within 0.5 cycles if derived from the same cDNA sample

3.  Moreover whatever the exact Ct value it must remain constant for the same samples within and between real time assays

4.  Finally to ensure accurate normalisation and gene expression given that different cDNA samples can vary slightly in Ct value ( due to quality differences as explained) on every real time assay GOI amplification data should always be normalised with housekeeping data derived from the same cDNA sample. In other words for different cDNA samples - treated untreated or just different biological replicates of both - in every assay you must amplify your gene of interest and your housekeeping gene from the same cDNA sample to ensure accurate normalisation for each data set in the assay 

https://www.researchgate.net/post/Any_advice_on_the_difference_in_the_Ct_value_of_reference_gene_in_treated_and_control_samples