Hi Maisarah, Genespring is a powerful tool to decipher differences when comparing two conditions using microarray data. But since it uses pure statistical tests everyone must remember rules in those types of analysis. When comparing two sets of BIOLOGICAL replicates, number of the replicates will allow you to raise some differential expression. in this case, 2 replicates is largely not enough and must start at 3, 5 is better. to avoid batch effects, all samples from both groups must also be treated by the same way and same time. the only difference must be biological.

all the best

fred

Hi Maisarah,

As Frederic mentions the most important issue is to increase de number of replicates and, in order to avoid batch effects, run them at the same time.

You could also reduce the fold change to 1.5 or even to 1.2 because the differences might be slight but hopefully significant after correcting for multiple testing. Finally you will have to validate your array results with RT-PCR to make sure that the differences in expression are consistent.

Good luck,

Maribel

Hi Maribel

I would put a particular attention when reducing the FC with raising of false negatives. In addition, probes used for GEP can work with different specificity and efficiency compared to primers designed for PCR. the best way is to design around the probe.

all the best

fred

Thank you Frederic Lepretre and Maribel Baldellou for the advice..

My lab usually prepared two replicates for statistical analysis in microarray, and ran by the experienced technical staff.. but since this time the problem is about technicalities, we will prepare new samples and run the replicates at the same time.

As we need to have some hints from the DEG, for now we use FC>2 and will validate some using qPCR. I will design the primers around the probe.

Thanks again!