Thanks Sean and John for your help.

Sean- I tried with relaxed stringency crietria Adj.Pvalues <0.05. Still no genes are satisfying this criteria.

But if just see my unadjusted Pvalues (unadjusted.P-values<0.01) they look good and I have 400 candidate genes that are differentially expressed. Can I consider the criteria of unadjusted P values? I am not a statistician so I am not very clear where to consider adjusted P-values and where to consider unadjusted ones? Can any one please explain me.

Below I am copying few lines of my top results. Why so much difference between adjusted P values and unadjusted P-values? Any help/suggestions would be much appreciated.

Many thanks,
R.

ID logFC AveExpr t P.Value adj.P.Val B

xx1_at -2.0682486 8.298777 -5.671953 4.754428e-05 0.4441129 1.16804549

xx3_at -1.1045124 7.838776 -5.446288 7.206374e-05 0.4441129 0.90168939

xx9_at -0.9933025 5.900082 -5.378236 8.180199e-05 0.4441129 0.81951230

xx5_at 0.5784688 5.979741 5.211694 1.118385e-04 0.4441129 0.61478979

xx2_at -1.1998221 8.423590 -5.174542 1.199786e-04 0.4441129 0.56842423

xx8_at -1.7810264 7.939280 -5.071211 1.460053e-04 0.4441129 0.43814027

xx4_at -0.4775558 2.965913 -5.026975 1.588722e-04 0.4441129 0.38177252

xx3_at 0.4783773 6.547145 4.917570 1.959800e-04 0.4441129 0.24084966

xx2_at -0.6317992 2.953137 -4.795892 2.479321e-04 0.4441129 0.08161861

xx7_at -0.5901271 3.606651 -4.743114 2.747013e-04 0.4441129 0.01174557

xx2_at -0.6615366 5.228286 -4.708753 2.937133e-04 0.4441129 -0.03400289

xx4_at -1.5438633 5.824030 -4.674656 3.139213e-04 0.4441129 -0.07960106

xx2_at 0.5362903 4.838442 4.613063 3.541284e-04 0.4441129 -0.16246898

If the adjusted p value is actually a False Discovery Rate, then requiring 0.01 is overly stringent.

It is certainly possible that you have real differences which are not reaching significance level because of your small sample size. Increasing replicates may help. But, also try some pre-filtering which I suggest in my answer below.