Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA


Autoři: Heejin Choi aff001;  Zhengpin Wang aff001;  Jurrien Dean aff001
Působiště autorů: Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD, United States of America aff001
Vyšlo v časopise: Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA. PLoS Genet 17(4): e1009485. doi:10.1371/journal.pgen.1009485
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009485

Souhrn

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

Klíčová slova:

Acrosomes – Germ cells – Sperm – Sperm head – Spermatids – Spermatogenesis – Testes – Vesicles


Zdroje

1. Hilz S, Modzelewski AJ, Cohen PE, Grimson A. The roles of microRNAs and siRNAs in mammalian spermatogenesis. Development. 2016;143(17):3061–73. Epub 2016/09/01. doi: 10.1242/dev.136721 27578177; PubMed Central PMCID: PMC5047671.

2. Sharma U, Sun F, Conine CC, Reichholf B, Kukreja S, Herzog VA, et al. Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm. Dev Cell. 2018;46(4):481–94 e6. Epub 2018/07/31. doi: 10.1016/j.devcel.2018.06.023 30057273; PubMed Central PMCID: PMC6103849.

3. Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N, et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature. 2006;442(7099):203–7. Epub 2006/06/06. doi: 10.1038/nature04916 16751777.

4. Girard A, Sachidanandam R, Hannon GJ, Carmell MA. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature. 2006;442(7099):199–202. Epub 2006/06/06. doi: 10.1038/nature04917 16751776.

5. Li XZ, Roy CK, Dong X, Bolcun-Filas E, Wang J, Han BW, et al. An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes. Mol Cell. 2013;50(1):67–81. Epub 2013/03/26. doi: 10.1016/j.molcel.2013.02.016 23523368; PubMed Central PMCID: PMC3671569.

6. Aravin AA, Sachidanandam R, Bourc’his D, Schaefer C, Pezic D, Toth KF, et al. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol Cell. 2008;31(6):785–99. Epub 2008/10/17. doi: 10.1016/j.molcel.2008.09.003 18922463; PubMed Central PMCID: PMC2730041.

7. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD. A distinct small RNA pathway silences selfish genetic elements in the germline. Science. 2006;313(5785):320–4. Epub 2006/07/01. doi: 10.1126/science.1129333 16809489.

8. Hartig JV, Tomari Y, Forstemann K. piRNAs—the ancient hunters of genome invaders. Genes Dev. 2007;21(14):1707–13. Epub 2007/07/20. doi: 10.1101/gad.1567007 17639076.

9. Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M, et al. DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes Dev. 2008;22(7):908–17. Epub 2008/04/03. doi: 10.1101/gad.1640708 18381894; PubMed Central PMCID: PMC2279202.

10. Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van den Elst H, et al. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell. 2007;129(1):69–82. Epub 2007/04/10. doi: 10.1016/j.cell.2007.03.026 17418787.

11. Batista PJ, Ruby JG, Claycomb JM, Chiang R, Fahlgren N, Kasschau KD, et al. PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans. Mol Cell. 2008;31(1):67–78. Epub 2008/06/24. doi: 10.1016/j.molcel.2008.06.002 18571452; PubMed Central PMCID: PMC2570341.

12. Das PP, Bagijn MP, Goldstein LD, Woolford JR, Lehrbach NJ, Sapetschnig A, et al. Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline. Mol Cell. 2008;31(1):79–90. Epub 2008/06/24. doi: 10.1016/j.molcel.2008.06.003 18571451; PubMed Central PMCID: PMC3353317.

13. Gainetdinov I, Colpan C, Arif A, Cecchini K, Zamore PD. A single mechanism of biogenesis, initiated and directed by PIWI proteins, explains piRNA production in most animals. Mol Cell. 2018;71(5):775–90 Epub 2018/09/08. doi: 10.1016/j.molcel.2018.08.007 30193099; PubMed Central PMCID: PMC6130920.

14. Reuter M, Berninger P, Chuma S, Shah H, Hosokawa M, Funaya C, et al. Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing. Nature. 2011;480(7376):264–7. Epub 2011/11/29. doi: 10.1038/nature10672 22121019.

15. Zheng K, Wang PJ. Blockade of pachytene piRNA biogenesis reveals a novel requirement for maintaining post-meiotic germline genome integrity. PLoS Genet. 2012;8(11):e1003038. Epub 2012/11/21. doi: 10.1371/journal.pgen.1003038 23166510; PubMed Central PMCID: PMC3499362.

16. Wasik KA, Tam OH, Knott SR, Falciatori I, Hammell M, Vagin VV, et al. RNF17 blocks promiscuous activity of PIWI proteins in mouse testes. Genes Dev. 2015;29(13):1403–15. Epub 2015/06/28. doi: 10.1101/gad.265215.115 26115953; PubMed Central PMCID: PMC4511215.

17. Castaneda J, Genzor P, van der Heijden GW, Sarkeshik A, Yates JR 3rd, Ingolia NT, et al. Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice. EMBO J. 2014;33(18):1999–2019. Epub 2014/07/27. doi: 10.15252/embj.201386855 25063675; PubMed Central PMCID: PMC4195769.

18. Gou LT, Dai P, Yang JH, Xue Y, Hu YP, Zhou Y, et al. Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis. Cell Res. 2014;24(6):680–700. Epub 2014/05/03. doi: 10.1038/cr.2014.41 24787618; PubMed Central PMCID: PMC4042167.

19. Goh WS, Falciatori I, Tam OH, Burgess R, Meikar O, Kotaja N, et al. piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis. Genes Dev. 2015;29(10):1032–44. Epub 2015/05/23. doi: 10.1101/gad.260455.115 25995188; PubMed Central PMCID: PMC4441051.

20. Zhang P, Kang JY, Gou LT, Wang J, Xue Y, Skogerboe G, et al. MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes. Cell Res. 2015;25(2):193–207. Epub 2015/01/15. doi: 10.1038/cr.2015.4 25582079; PubMed Central PMCID: PMC4650574.

21. Vourekas A, Zheng Q, Alexiou P, Maragkakis M, Kirino Y, Gregory BD, et al. Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis. Nat Struct Mol Biol. 2012;19(8):773–81. Epub 2012/07/31. doi: 10.1038/nsmb.2347 22842725; PubMed Central PMCID: PMC3414646.

22. Homolka D, Pandey RR, Goriaux C, Brasset E, Vaury C, Sachidanandam R, et al. PIWI slicing and RNA elements in precursors instruct directional primary piRNA biogenesis. Cell Rep. 2015;12(3):418–28. Epub 2015/07/15. doi: 10.1016/j.celrep.2015.06.030 26166577.

23. Wu PH, Fu Y, Cecchini K, Ozata DM, Arif A, Yu T, et al. The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility. Nat Genet. 2020;52(7):728–39. Epub 2020/07/01. doi: 10.1038/s41588-020-0657-7 32601478; PubMed Central PMCID: PMC7383350.

24. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil J, editors. The Physiology of Reproduction. 2 ed. New York: Raven Press; 1994. p. 189–317.

25. Leblond CP, Clermont Y. Spermiogenesis of rat, mouse, hamster and guinea pig as revealed by the periodic acid-fuchsin sulfurous acid technique. Am J Anat. 1952;90(2):167–215. Epub 1952/03/01. doi: 10.1002/aja.1000900202 14923625.

26. Abou-Haila A, Tulsiani DR. Mammalian sperm acrosome: formation, contents, and function. Arch Biochem Biophys. 2000;379(2):173–82. Epub 2000/07/19. doi: 10.1006/abbi.2000.1880 10898932.

27. Kierszenbaum AL, Tres LL. The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head. Arch Histol Cytol. 2004;67(4):271–84. Epub 2005/02/11. doi: 10.1679/aohc.67.271 15700535.

28. Buffone MG, Foster JA, Gerton GL. The role of the acrosomal matrix in fertilization. Int J Dev Biol. 2008;52(5–6):511–22. Epub 2008/07/24. doi: 10.1387/ijdb.072532mb 18649264.

29. Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA, et al. Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proc Natl Acad Sci U S A. 2011;108(12):4892–6. Epub 2011/03/09. doi: 10.1073/pnas.1018202108 21383182; PubMed Central PMCID: PMC3064341.

30. Berruti G, Paiardi C. Acrosome biogenesis: Revisiting old questions to yield new insights. Spermatogenesis. 2011;1(2):95–8. Epub 2012/02/10. doi: 10.4161/spmg.1.2.16820 22319656; PubMed Central PMCID: PMC3271650.

31. Zhou L, Canagarajah B, Zhao Y, Baibakov B, Tokuhiro K, Maric D, et al. BTBD18 regulates a subset of piRNA-generating loci through transcription elongation in mice. Dev Cell. 2017;40(5):453–66 Epub 2017/03/16. doi: 10.1016/j.devcel.2017.02.007 28292424.

32. Bath ML. Inhibition of in vitro fertilizing capacity of cryopreserved mouse sperm by factors released by damaged sperm, and stimulation by glutathione. PLoS One. 2010;5(2):e9387. Epub 2010/03/03. doi: 10.1371/journal.pone.0009387 20195370; PubMed Central PMCID: PMC2827551.

33. Takeo T, Nakagata N. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biol Reprod. 2011;85(5):1066–72. Epub 2011/07/23. doi: 10.1095/biolreprod.111.092536 21778138.

34. Turner KJ, Sharpe RM, Gaughan J, Millar MR, Foster PM, Saunders PT. Expression cloning of a rat testicular transcript abundant in germ cells, which contains two leucine zipper motifs. Biol Reprod. 1997;57(5):1223–32. Epub 1997/11/22. doi: 10.1095/biolreprod57.5.1223 9369191.

35. Brohmann H, Pinnecke S, HoyerFender S. Identification and characterization of new cDNAs encoding outer dense fiber proteins of rat sperm. J Biol Chem. 1997;272(15):10327–32. WOS:A1997WU03900104. doi: 10.1074/jbc.272.15.10327 9092585

36. Tarnasky H, Cheng M, Ou Y, Thundathil JC, Oko R, van der Hoorn FA. Gene trap mutation of murine outer dense fiber protein-2 gene can result in sperm tail abnormalities in mice with high percentage chimaerism. Bmc Dev Biol. 2010;10:67. Epub 2010/06/17. doi: 10.1186/1471-213X-10-67 20550699; PubMed Central PMCID: PMC2894780.

37. Ito C, Yamatoya K, Yoshida K, Fujimura L, Hatano M, Miyado K, et al. Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy. Cell Tissue Res. 2013;352(3):739–50. Epub 2013/04/09. doi: 10.1007/s00441-013-1605-y 23564009.

38. Roqueta-Rivera M, Abbott TL, Sivaguru M, Hess RA, Nakamura MT. Deficiency in the omega-3 fatty acid pathway results in failure of acrosome biogenesis in mice. Biol Reprod. 2011;85(4):721–32. Epub 2011/06/10. doi: 10.1095/biolreprod.110.089524 21653892.

39. Kanemori Y, Koga Y, Sudo M, Kang W, Kashiwabara S, Ikawa M, et al. Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse. Proc Natl Acad Sci U S A. 2016;113(26):E3696–705. Epub 2016/06/16. doi: 10.1073/pnas.1522333113 27303034; PubMed Central PMCID: PMC4932935.

40. Kierszenbaum AL, Rivkin E, Tres LL. Acroplaxome, an F-actin-keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head. Mol Biol Cell. 2003;14(11):4628–40. Epub 2003/10/11. doi: 10.1091/mbc.e03-04-0226 14551252; PubMed Central PMCID: PMC266778.

41. Nebel BR, Amarose AP, Hacket EM. Calendar of gametogenic development in the prepuberal male mouse. Science. 1961;134(3482):832–3. Epub 1961/09/22. doi: 10.1126/science.134.3482.832 13728067.

42. Han F, Liu C, Zhang L, Chen M, Zhou Y, Qin Y, et al. Globozoospermia and lack of acrosome formation in GM130-deficient mice. Cell Death Dis. 2017;8(1):e2532. Epub 2017/01/06. doi: 10.1038/cddis.2016.414 28055014; PubMed Central PMCID: PMC5386352.

43. Roy E, Bruyere J, Flamant P, Bigou S, Ausseil J, Vitry S, et al. GM130 gain-of-function induces cell pathology in a model of lysosomal storage disease. Hum Mol Genet. 2012;21(7):1481–95. Epub 2011/12/14. doi: 10.1093/hmg/ddr584 22156940.

44. Deng W, Lin H. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev Cell. 2002;2(6):819–30. Epub 2002/06/14. doi: 10.1016/s1534-5807(02)00165-x 12062093.

45. Grivna ST, Beyret E, Wang Z, Lin H. A novel class of small RNAs in mouse spermatogenic cells. Genes Dev. 2006;20(13):1709–14. Epub 2006/06/13. doi: 10.1101/gad.1434406 16766680; PubMed Central PMCID: PMC1522066.

46. Ding D, Liu J, Midic U, Wu Y, Dong K, Melnick A, et al. TDRD5 binds piRNA precursors and selectively enhances pachytene piRNA processing in mice. Nat Commun. 2018;9(1):127. Epub 2018/01/11. doi: 10.1038/s41467-017-02622-w 29317670; PubMed Central PMCID: PMC5760656.

47. Xu M, You Y, Hunsicker P, Hori T, Small C, Griswold MD, et al. Mice deficient for a small cluster of Piwi-interacting RNAs implicate Piwi-interacting RNAs in transposon control. Biol Reprod. 2008;79(1):51–7. Epub 2008/04/11. doi: 10.1095/biolreprod.108.068072 18401007.

48. Han BW, Wang W, Zamore PD, Weng Z. piPipes: a set of pipelines for piRNA and transposon analysis via small RNA-seq, RNA-seq, degradome- and CAGE-seq, ChIP-seq and genomic DNA sequencing. Bioinformatics. 2015;31(4):593–5. Epub 2014/10/25. doi: 10.1093/bioinformatics/btu647 25342065; PubMed Central PMCID: PMC4325541.

49. Bartel DP. Metazoan MicroRNAs. Cell. 2018;173(1):20–51. WOS:000428234200006. doi: 10.1016/j.cell.2018.03.006 29570994

50. Dai P, Wang X, Gou LT, Li ZT, Wen Z, Chen ZG, et al. A translation-activating function of MIWI/piRNA during mouse spermiogenesis. Cell. 2019;179(7):1566–81. doi: 10.1016/j.cell.2019.11.022 WOS:000502546200014. 31835033

51. Aravin AA. Pachytene piRNAs as beneficial regulators or a defense system gone rogue. Nature Genetics. 2020;52(7):644–5. doi: 10.1038/s41588-020-0656-8 WOS:000544170300003. 32601474

52. Wang H, Wan H, Li X, Liu W, Chen Q, Wang Y, et al. Atg7 is required for acrosome biogenesis during spermatogenesis in mice. Cell Res. 2014;24(7):852–69. Epub 2014/05/24. doi: 10.1038/cr.2014.70 24853953; PubMed Central PMCID: PMC4085765.

53. Xiao N, Kam C, Shen C, Jin W, Wang J, Lee KM, et al. PICK1 deficiency causes male infertility in mice by disrupting acrosome formation. J Clin Invest. 2009;119(4):802–12. Epub 2009/03/05. doi: 10.1172/JCI36230 19258705; PubMed Central PMCID: PMC2662547.

54. Lin YN, Roy A, Yan W, Burns KH, Matzuk MM. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis. Mol Cell Biol. 2007;27(19):6794–805. Epub 2007/08/01. doi: 10.1128/MCB.01029-07 17664285; PubMed Central PMCID: PMC2099232.

55. Funaki T, Kon S, Tanabe K, Natsume W, Sato S, Shimizu T, et al. The Arf GAP SMAP2 is necessary for organized vesicle budding from the trans-Golgi network and subsequent acrosome formation in spermiogenesis. Mol Biol Cell. 2013;24(17):2633–44. Epub 2013/07/19. doi: 10.1091/mbc.E13-05-0234 23864717; PubMed Central PMCID: PMC3756916.

56. Tardif S, Guyonnet B, Cormier N, Cornwall GA. Alteration in the processing of the ACRBP/sp32 protein and sperm head/acrosome malformations in proprotein convertase 4 (PCSK4) null mice. Mol Hum Reprod. 2012;18(6):298–307. Epub 2012/02/24. doi: 10.1093/molehr/gas009 22357636; PubMed Central PMCID: PMC3358042.

57. Yan W, Ma L, Burns KH, Matzuk MM. Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice. Proc Natl Acad Sci U S A. 2004;101(20):7793–8. Epub 2004/05/12. doi: 10.1073/pnas.0308025101 15136734; PubMed Central PMCID: PMC419685.

58. Pierre V, Martinez G, Coutton C, Delaroche J, Yassine S, Novella C, et al. Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus. Development. 2012;139(16):2955–65. Epub 2012/07/06. doi: 10.1242/dev.077982 22764053.

59. Kang-Decker N, Mantchev GT, Juneja SC, McNiven MA, van Deursen JM. Lack of acrosome formation in Hrb-deficient mice. Science. 2001;294(5546):1531–3. Epub 2001/11/17. doi: 10.1126/science.1063665 11711676.

60. Gerton GL, Millette CF. Generation of flagella by cultured mouse spermatids. J Cell Biol. 1984;98(2):619–28. Epub 1984/02/01. doi: 10.1083/jcb.98.2.619 6363426; PubMed Central PMCID: PMC2113102.

61. Russell LD, Weiss T, Goh JC, Curl JL. The effect of submandibular gland removal on testicular and epididymal parameters. Tissue Cell. 1990;22(3):263–8. Epub 1990/01/01. doi: 10.1016/0040-8166(90)90001-p 2237906.

62. Dia F, Strange T, Liang J, Hamilton J, Berkowitz KM. Preparation of meiotic chromosome spreads from mouse spermatocytes. J Vis Exp. 2017;(129). Epub 2017/12/30. doi: 10.3791/55378 29286440; PubMed Central PMCID: PMC5755458.

63. Roovers EF, Rosenkranz D, Mahdipour M, Han CT, He N, Chuva de Sousa Lopes SM, et al. Piwi proteins and piRNAs in mammalian oocytes and early embryos. Cell Rep. 2015;10(12):2069–82. Epub 2015/03/31. doi: 10.1016/j.celrep.2015.02.062 25818294.

64. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–60. Epub 2015/03/10. doi: 10.1038/nmeth.3317 25751142; PubMed Central PMCID: PMC4655817.

65. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–9. Epub 2009/06/10. doi: 10.1093/bioinformatics/btp352 19505943; PubMed Central PMCID: PMC2723002.

66. Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011;27(21):2987–93. Epub 2011/09/10. doi: 10.1093/bioinformatics/btr509 21903627; PubMed Central PMCID: PMC3198575.

67. Liao Y, Smyth GK, Shi W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013;41(10):e108. Epub 2013/04/06. doi: 10.1093/nar/gkt214 23558742; PubMed Central PMCID: PMC3664803.

68. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. Epub 2014/12/18. doi: 10.1186/s13059-014-0550-8 25516281; PubMed Central PMCID: PMC4302049.

69. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14(4):417–9. Epub 2017/03/07. doi: 10.1038/nmeth.4197 28263959; PubMed Central PMCID: PMC5600148.

70. Walker M, Billings T, Baker CL, Powers N, Tian H, Saxl RL, et al. Affinity-seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage. Epigenetics Chromatin. 2015;8:31. Epub 2015/09/10. doi: 10.1186/s13072-015-0024-6 26351520; PubMed Central PMCID: PMC4562113.

71. Bao W, Kojima KK, Kohany O. Repbase update, a database of repetitive elements in eukaryotic genomes. Mob DNA. 2015;6:11. Epub 2015/06/06. doi: 10.1186/s13100-015-0041-9 26045719; PubMed Central PMCID: PMC4455052.

72. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9. Epub 2012/03/06. doi: 10.1038/nmeth.1923 22388286; PubMed Central PMCID: PMC3322381.

73. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402–8. Epub 2002/02/16. doi: 10.1006/meth.2001.1262 11846609.


Článek vyšel v časopise

PLOS Genetics


2021 Číslo 4
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

Jak na primární i sekundární osteoporózu − prakticky a v kostce
nový kurz
Autoři: MUDr. Jan Rosa

Léčba roztroušené sklerózy

Důležitost adherence při depresivním onemocnění
Autoři: MUDr. Eliška Bartečková, Ph.D.

Koncepce osteologické péče pro gynekology a praktické lékaře
Autoři: MUDr. František Šenk

Sekvenční léčba schizofrenie
Autoři: MUDr. Jana Hořínková, Ph.D.

Všechny kurzy
Kurzy Podcasty Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se