[The effect of the split of sperm nuclei on the yield of human sperm RNA].
OBJECTIVE
To study the effect of the split of sperm nuclei on the yield of RNA from human sperm.


METHODS
Human sperm were purified by two sequential centrifugations through 40:80 discontinuous gradients of Percoll. Human leukocytes separated from peripheral blood were used as the control. Total RNAs from purified sperm and leukocytes were extracted with both TRIzol and RNeasy Kit. The RNAs from equal number of cells were reverse-transcribed, and quantified by the levels of beta-ACTIN mRNA, which were evaluated by real time polymerase chain reaction.


RESULTS
TRIzol failed to digest majority of sperm nuclei even the incubation time was prolonged to 1 h, while no sperm nucleus was found under the light microscope after 1 min digestion with RLT buffer of the RNeasy Kit. Both reagents can digest the nuclei of human leukocytes well. The amount of RNA per 10(6) sperms isolated with RNeasy Kit (149.8+/-24.5 ng) was 4-fold higher (P=0.01) than that extracted with TRIzol (35.5+/-4.0 ng per 10(6) spermatozoa; n=3). The similar yields of the leukocyte RNAs extracted with RNeasy Kit and TRIzol [(765.5+/-229.8) and (958.8+/-201.0) ng per 10(6) cells respectively; n=3, P=0.168] excluded the possibility of different efficacy of these two reagents in RNA isolation.


CONCLUSIONS
The split of sperm nuclei is important to the yield of RNA in the human sperm RNA extraction. The nucleus may be the major area for human sperm RNA repositories.
Li
Hong-gang
HG
Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030 People's Republic of China.
Ding
Xiao-fang
XF
Xiong
Cheng-liang
CL
D002467:Cell Nucleus / Q000737:chemistry*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis; D013094:Spermatozoa / Q000737:chemistry*
D012333:RNA, Messenger; D012313:RNA