• You cannot compensate for biological replicates by using more technical replicates
• In essence, your first experiment should include 3 technical replicates derived from 1 biological replicate (for which you determine replicate mean for calculation purposes
• What is more, your technical replicates for a particular conditions should be no more than x +/- 0.5 cycles to determine a valid mean
• If having determined a vald mean from concordant technical replicates you see an interesting affect on gene expression for a particular treatment you then repeat the experiment and independently verify with means derived from new technical replicates derived from a new biological replicate
• That way you can be sure that if you see a REPRODUCIBLE effect, it is not some weird artefact linked to stasis conditions of the first biological replicate but rather is seen in 1 or more independent biological replicates; in other words the effect you are seeing is a real biological phenomenon and not a technical artifact
• If you simply include additional technical replicates either in the same experiment or in a repeat experiment you can never be sure that the effect you see is something anomalous with your first biological replicate
• Biological replicates therefore are designed to test whether gene expression is a function of treatment whereas technical replicates are designed to quantify that effect if seen

• You cannot compensate for biological replicates by using more technical replicates
• In essence, your first experiment should include 3 technical replicates derived from 1 biological replicate (for which you determine replicate mean for calculation purposes
• What is more, your technical replicates for a particular conditions should be no more than x +/- 0.5 cycles to determine a valid mean
• If having determined a vald mean from concordant technical replicates you see an interesting affect on gene expression for a particular treatment you then repeat the experiment and independently verify with means derived from new technical replicates derived from a new biological replicate
• That way you can be sure that if you see a REPRODUCIBLE effect, it is not some weird artefact linked to stasis conditions of the first biological replicate but rather is seen in 1 or more independent biological replicates; in other words the effect you are seeing is a real biological phenomenon and not a technical artifact
• If you simply include additional technical replicates either in the same experiment or in a repeat experiment you can never be sure that the effect you see is something anomalous with your first biological replicate
• Biological replicates therefore are designed to test whether gene expression is a function of treatment whereas technical replicates are designed to quantify that effect if seen

Dear Dr. Laurence Stuart Dawkins-Hall , I thank you very much for such a kind reply. It made my concepts clear.

Laurence has excellent points, to add it to, with n=1 you wouldn't be able to perform inferential statistics due to n-1 degrees of freedom being 0 degrees of freedom (df) (some scenarios are df=n-k and other). Think about it, with n=1 you have no basis to estimate variance and standard deviation. Without the standard deviation, how can your calculate z/t-stastistic? So you would have hard time doing unpaired t-tests and ANOVAs with you data.

Good add on Can!

Dear Dr./Sir Can Kiessling , It is a valid point and I thank you very much.

Dear Dr. Laurence Stuart Dawkins-Hall , I am sorry if I am bothering you again.

In case of low total RNA leading (500ng in 10ul) to low cDNA concentration (500ng in 20ul). Is it fine to pool biological replicates and then use triplicates?

I have made dilution of cDNA to 15ng/ul and used 1ul in a conventional PCR to check absence/presence of transcript for a certain gene. My HKG (B-Actin) showed bright bands for all treatments (01 biological replicate each). I didn't use technicals here.

Would it be right if i pool my biological replicates and make 3 technicals in real time pcr for gene expression?

Dr. Can Kiessling please add too.

Yes it is provided the RNA is derived from the same cells treated in the same way

But then be sure to repeat your essay