Hi Zahid,

You do not necessarily need to prepare a standard curve, because if you want to compare the expression of mRNA you have to calculate the relative expression (the change in expression amongst treated and untreated samples). You cannot use the houskeeping to directly compare expression because they are different genes, expressed in different amounts. I suggest you to perform this type of experiment:

Gene A: housekeeping

Gene B: Target

Then you measure mRNA (without standard curve, just raw reading) in two conditions: cells untreated with RNAi (B1) and cells treated (B2). In this way you will see a difference in the Cq of the samples, because (in theory) knocked cells should have less mRNA, so in B2 you should have a higher Cq, meaning later amplification of target RNA. 

But here is where you need the housekeeping gene. You can't be sure that in the treated and untreated cells the conditions are the same, maybe you saw a difference just for chance or because of an error in the experimental procedure.

So, you use the housekeeping gene to normalize the values. Its expression should never change across samples, it should always be present. So you can use the hk in the treated and unreated samples as a reference to really measure your mRNA.

I'll try to explain: You have a Cq of 30 of B2 and a Cq of 30 of A2 (the housekeeping) and then you have a Cq of 20 of B1 and a Cq of 20 of B1. It seems target mRNA is more expressed in B1 (Cq is lower, there is more amplification) but in reality in condition 1 there is also more housekeeping gene. But the housekeeping should be hydentical! If there is less hk  in condition 2 it means that maybe you had less template, or the extraction was worst.

You compare the target amount to the housekeeping amount to reduce errors, basically.

In the end you obtain normalized values for each experiment and you can use your untreated cells to compare the expression of treated cells.

Almost all qPCR instruments can do Relative quantification automatically (I did it with Eppendorf and Applied Biosystems). Otherwise there are programs like QBASE+

It is a bit complicated to explain, you can see the attachments or search for qpcr relative quantification assays.

https://www.thermofisher.com/it/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html

http://relative.gene-quantification.info/

Hi Zahid,

You do not necessarily need to prepare a standard curve, because if you want to compare the expression of mRNA you have to calculate the relative expression (the change in expression amongst treated and untreated samples). You cannot use the houskeeping to directly compare expression because they are different genes, expressed in different amounts. I suggest you to perform this type of experiment:

Gene A: housekeeping

Gene B: Target

Then you measure mRNA (without standard curve, just raw reading) in two conditions: cells untreated with RNAi (B1) and cells treated (B2). In this way you will see a difference in the Cq of the samples, because (in theory) knocked cells should have less mRNA, so in B2 you should have a higher Cq, meaning later amplification of target RNA. 

But here is where you need the housekeeping gene. You can't be sure that in the treated and untreated cells the conditions are the same, maybe you saw a difference just for chance or because of an error in the experimental procedure.

So, you use the housekeeping gene to normalize the values. Its expression should never change across samples, it should always be present. So you can use the hk in the treated and unreated samples as a reference to really measure your mRNA.

I'll try to explain: You have a Cq of 30 of B2 and a Cq of 30 of A2 (the housekeeping) and then you have a Cq of 20 of B1 and a Cq of 20 of B1. It seems target mRNA is more expressed in B1 (Cq is lower, there is more amplification) but in reality in condition 1 there is also more housekeeping gene. But the housekeeping should be hydentical! If there is less hk  in condition 2 it means that maybe you had less template, or the extraction was worst.

You compare the target amount to the housekeeping amount to reduce errors, basically.

In the end you obtain normalized values for each experiment and you can use your untreated cells to compare the expression of treated cells.

Almost all qPCR instruments can do Relative quantification automatically (I did it with Eppendorf and Applied Biosystems). Otherwise there are programs like QBASE+

It is a bit complicated to explain, you can see the attachments or search for qpcr relative quantification assays.

https://www.thermofisher.com/it/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html

http://relative.gene-quantification.info/

Umberto's answer is very clear and gives a good example of a standard relative quantification experiment. The only thing that I would recommend is that you measure more than one housekeeping gene, just to be sure that you are choosing one which has stable expression under your experimental conditions, certainly for the first attempt at this. Once you've identified the most robust housekeeping gene, you only need to measure that one in future. 

As Umberto points out, discrepancy in housekeeping genes could be due to the amount of loading. In order to control for this, I always load equal concentrations of RNA (5ng/ul) so that any fluctuations in housekeeping genes are flagged up and I can assess whether my experimental conditions have affected their expression. If they have, I discard that housekeeping gene and use a different one.

I think the answers of Umberto and Mark are well explained, however, I want to add some more information regarding the use of housekeeping genes for normalization. The concept that housekeeping genes expression is consistent is an old story. Now, there are several studies which report that the expression of housekeeping genes doesn't stay stable in different experimental conditions. Thus, validation of housekeeping genes is a necessary step to avoid erroneous results. I would like to suggest that first, you should try to search out literature about the validation of housekeeping genes in the species or tissue or cells in which you are going to measure the expression, perhaps you can find some of the housekeeping genes which have already been validated in your area of interest. By doing this, you don't have to validate the housekeeping genes by yourself and you can choose the stable one from already published data. Also, you need to add the reference of that paper when you mention about that gene so that the reviewers can know that it was already validated. 

I agree with Mohammed on this one point

Look to programs like Gene Norm and Normfinder for selecting the most stable housekeeping genes from a panel of housekeeping genes (evaluate > 6; Select 2-3) in the context of your tissue/ treatments