Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos-0

<p><b>Copyright information:</b></p><p>Taken from "Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/58</p><p>BMC Developmental Biology 2007;7():58-58.</p><p>Published online 31 May 2007</p><p>PMCID:PMC1896162.</p><p></p>DH 219 bp, H2A 83 bp, PGK1 126 bp, S18 149 bp, UBC 186 bp). 100 bp = 100 base pair ladder

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos-3

<p><b>Copyright information:</b></p><p>Taken from "Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/58</p><p>BMC Developmental Biology 2007;7():58-58.</p><p>Published online 31 May 2007</p><p>PMCID:PMC1896162.</p><p></p>DH 219 bp, H2A 83 bp, PGK1 126 bp, S18 149 bp, UBC 186 bp). 100 bp = 100 base pair ladder

Expression of NANOG and eGFP in mouse testes sections as detected by immunohistochemistry.

<p>(A–C) Expression of NANOG in mouse testis at different stages of spermatogenesis; Roman figures in seminiferous tubules represent the stage of the epithelial cycle; (D) Negative control section from which the primary antibody was omitted in the staining procedure; (E) Expression of eGFP in testis sections of a <i>Nanog</i> eGFP reporter; (F) eGFP staining on testis sections of a non-transgenic mouse, which served as a negative control for the staining in figures E. Abbreviations: AS =  type A spermatogonia, P =  Pachytene spermatocyte, T = Spermatid, ET =  elongating spermatids, S =  Sertoli cell, * =  non-specific binding to interstitial cells. Sections displayed in panels A–D were fixed in Bouins fixative; sections in panels E and F were fixed in formalin. Formalin fixation interfered with immunostaining of the seminiferous tubules.</p

Antibodies used for immuno applications.

<p>AA =  Amino Acids, IHC =  immunohistochemistry, IF =  immunofluorescence, IB = immunoblotting.</p

Immunoblot analysis.

<p>Composite image of immunoblot results for NANOG on lysates of mouse ES cells and dog and pig testes. On the right is a Benchmark protein ladder (Invitrogen) and the indicated molecular weights for each band. Abbreviations: mES =  mouse embryonic stem cells, <i>Cf</i> =  <i>Canis familiaris</i>, <i>Ss</i> =  <i>Sus scrofa</i>.</p

Expression of NANOG in pig testis as determined by immunofluorescence.

<p>(A–C) NANOG expression in differentiating male germ cells; (D–F) high magnification image of NANOG expression in pachytene spermatocytes; (G–I) image of section in which the primary antibody was replaced by blocking solution, to control for non-specific binding of the secondary antibody; (J–K) representative high magnification image of testis section that was incubated with a rabbit IgG isotype control, to control for non-specific binding of the primary antibody. Dashed lines in panels A–C and G–I mark the boundaries of the tubules.</p

Primers used for RT-PCR and sequence analysis.

<p>F =  forward primer, R =  reverse primer. Ta =  annealing temperature used in PCR reaction.</p

Expression of <i>Nanog</i> in testis of various mammalian species as determined by RT-PCR.

<p>(A) Expression of <i>Nanog</i> in mouse testis of the Black 6 strain (BL/6) and the SV129 strain (SV129) (B) Expression of <i>eGFP</i> in testis of a TNG mouse; (C) Expression of <i>NANOG</i> in dog and pig testes; (D) Expression of <i>NANOG</i> in human testis; in all RT-PCR experiments, amplicons were of the expected sizes (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010987#pone-0010987-t002" target="_blank">Table 2</a>) and products were identified by sequence analysis. Abbreviations: TNG =  testis cDNA from <i>Nanog</i> eGFP reporter mouse; <i>Cf</i> =  <i>Canis familiaris</i>, <i>Ss</i> =  <i>Sus scrofa</i>, <i>Hs</i> =  <i>Homo sapiens</i>; +  = cDNA synthesis reaction performed with reverse transcriptase, −  =  minus reverse transcriptase control.</p

Expression of NANOG in human testis as determined by immunofluorescence in paraffin-embedded sections.

<p>(A–C) NANOG expression in differentiating male germ cells; dashed lines mark the boundaries of tubules.</p

Y-chromomal sequence variation diagnostic for indicine and Y1 and Y2 taurine haplotypes.

*<p>Corresponding to the <i>Bfa</i>I site in banteng.</p><p>Database entries and the numbering refer to sequences from taurine cattle.</p