5% of the other tissues may be equal to or more than zttestis Th

5% of the other tissues may be equal to or more than zttestis. The following five expression levels were defined accordingly. A for absent of a gene in t if the ori ginal Affymetrix PA call value is A. P for present if the Affymetrix PA call value is P. HP for highly present with zt z1. MS for multi tissue specific with zt z2 be cause a gene with such an expression value may only expressed in several tissues. SP for specific with zt z3 for tissue t and zi z2 for all the other tissues. The most specific expression level was assigned to a gene if it satisfying more than one level. For example, a gene considered SP must also satisfy the MS condition, but we labeled it as SP. We considered five datasets as five voters. We determined the mRNA expression level by a ballot.
The expression level that was supported by the highest number of votes was assigned to the gene in the specified tissue. However, if two expression levels were both supported by the high est number of votes, the less specific level was assigned. For example, MS was assigned to a gene if both MS and SP were supported by 2 votes. Animal use Male CD1 mice were purchased from Vital p38 MAP Kinase inhibitor River La boratories and maintained in the Experiment Animal Center, Chinese Academy of Sciences. Animal use was approved by the Animal Re search Committee of the Institute of Zoology, Chinese Academy of Sciences. Protocols for animal sacrifice and tissue harvesting were in accord ance with the NIH Guide for the Care and Use of La boratory Animals. All the mice were euthanized by cervical dislocation before tissue harvesting.
Isolation of different type germ cells 8 dpp and 17 dpp mice were used for isolating type A spermatogonia and pachytene spermatocytes, respectively. Meanwhile, adult mice were used selelck kinase inhibitor for isolating round spermatids and elonged sper matids. The seminiferous tubules were isolated from decapsulated testes and digested into cell suspen sion with collagenase and trypsin. The purity of four types of germ cells all exceeded 90% using the unit grav ity sedimentation procedure in 2 4% BSA medium as described before. The purity of cells was vali dated based on morphological evaluation and confirmed by RT PCR of reported marker genes expressed in differ ent germ cells. RNA isolation and RT PCR Total RNAs were extracted from mouse testes at differ ent developmental stages and other tissues using TRIzol solution. 2 ug RNA was reverse transcribed into cDNA by reverse transcriptase. The primers used for PCR are listed in Table 4. PCR reactions were conducted following stand protocol. Briefly, the reactions were started at 94 C for 3 min, and went through 27 cycles with denaturing at 94 C for 30 sec, annealing 60 C for 30 sec, and elongation at 72 C for 40 sec.

5% of the other tissues may be equal to or more than zttestis Th