Actually I am confused about the meaning of biological replications ,at the moment I am trying to design a method and I want to study the gene expression .I want to use one animal under specific condition .So my question is
If I want to do 4 biological replications, should I extract the RNA from the whole animal (homogenize the whole animal and take 4 isolation )
Or should I extract RNA from different 4 organs from the same animal?
Or should I use 4 animal under the same conditions?
i am confused about the meaning of biological replications
So am I, and I think the distinction biological vs technical doesn't make any sense. You should randomly replicate the level(s) of variation that you want to estimate, whether you call that level "biological" or "technical" doesn't change anything.
If you are interesting in the variation between mice within a certain breed, than you should randomly replicate mice within that breed. If you are also interested in the variation between different explants from that same organ, you should randomly split the organ and process the different bits independently. Or again, you might be interested in seeing how different library preparation protocols performs, so you could replicate library preps within the same RNA extraction.
In this experiment, does it matters which level is biological and which is technical? I don't think so, what matters is the question you are interested at.
I am usually confident about technical errors on a properly calibrated instrument or with a proven technique (unless you are developping a new technique or you are trying to see why an experiment fails repeatedly). Now, why should we bother doing biological replicates?
Let's suppose you are doing an experiment with two conditions A and B. You want to find if some particular RNA is overexpressed in A. You do your experiment and you find in A it is twice more expressed than in B. What can you conclude?
Not a lot. Maybe the levels of your RNA fluctuate a lot naturally. Maybe your animal had a hidden condition affecting it. If you do biological replicates, you can determine if you have similar levels of expression across individuals. You can then determine if the difference between conditions is mainly due to normal fluctuations between individuals or to a bona fide influence of your conditions.
EDIT: in one of my experiments, I was comparing two groups of mice: in one a two-parts construct (driven by TetO TetA) was active, in another it was missing one half and should have been very weakly expressed.. The experiments gave weird results, because in my inactive construct group, some mice expressed my transgene ten times more than others. They all had one particular half of the transgene, which turned out to have a faint basal expression due to an unlucky insertion in the genome.
If I had tested only a couple of mice, or only mice with one particular half of my transgene, I would have concluded everything was fine: my controls were a lot weaker than my mice with the active transgene. However, the low basal expression was also found in my mice with the full construct, and was enough to invalidate my results.
Biological replicates are parallel measurements of biologically distinct samples that capture random biological variation, which may itself be a subject of study or a noise source. Technical replicates are repeated measurements of the same sample that represent independent measures of the random noise associated with protocols or equipment. For biologically distinct conditions, averaging technical replicates can limit the impact of measurement error, but taking additional biological replicates is often preferable for improving the efficiency of statistical testing.