Biological replicates for cell lines

Question

[blixxed]

Hi

I would like to run a whole genome microarray experiment. I have a clonal cell line (the entire cell line derived from one cell) and I would like to knock down my gene of interest in this cell line using one of two siRNAs that target this gene.

To have three vs. three biological replicates, should I use the same clonal cell line and knockdown my gene using the same siRNA, then split that into 3? For control I could use a non-targeting siRNA on the same clonal cell line, then split into 3 and call these "biological replicates" OR is it better to use a pooled parental cell line and do the knockdowns in this cell line instead of using a clonal.. same anti-gene siRNA and non-targeting siRNA in 3 different wells?

[blixxed]

Thanks for the responses guys

I forgot to mention that I have 6 clonal cell lines from the same parent cell line, not one. Would you then recommend I use 3 of these clones as biological replicate "tests" and 3 as "controls"?

My main issue with using clonal cells is precisely the fact that they come from the same cell. If a gene knockdown effect in one of these clones is spectacularly good or bad, then this may not actually be something normal but due to that particular cell being special in some may. I thought using a pooled cell line with a mixture of cells from the same cell line would sort out any variation that may exist between the cells. I like the idea of using different passages though.

Answer 1

[Zhilong Jia]

Biological replicates: measure a quantity from difference sources under the same conditions. Tumors from 5 different people with lung cancer may show similar gene expression patterns. These replicates are useful to show what is similar in your replicates and how they are different from a different set of conditions (ie. treated, normal).

Technical replicates: measure quantity from 1 source. This measures the reproducibility of the results. The differences are based only on technical issues in the measurement. (I weigh myself three times, do I get different weights? How different?)

ref: http://wiki.answers.com/Q/What_is_defference_between_Biological_replicates_and_technical_replicates

Seems two clonal cell lines derived from one cell using same operation after primary culture are better.

To be honest, I'm not familiar with whole genome microarray experiment.

Answer 2

[Devon Ryan]

In a perfect world, you'd have multiple clonal cell-lines from similar starting cells. We do not, however, live in a perfect world. Realistically, you just want to get a grip on how variable your results might be if you were to repeat the experiment again. With a cell-line, you might be best off just using different passage numbers (assuming the line can be passaged) for the different replicates, since that'll at least guard against a random passage-number effect.