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2002, Number 1

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Bioquimia 2002; 27 (1)

Quantification of the genic expression from a limited number of cells through RT-PCR in real time

Bonilla E, Párraga M, López LA, Escolar F, Mazo J

Language: Spanish
References: 10
Page: 2-6
PDF size: 363.91 Kb.


ABSTRACT

Quantification of gene expression by classical methods requiresamounts of mRNA difficult to obtain when the number ofexperimental samples is limited. Recently, a new detectiontechnology that facilitates real-time detection of specificamplification product during PCR has been introduced(LigthCycler™ Roche). In this study, we have used thistechnology to analyze the expression of single genes in fetalmouse oocytes. RT-PCR was performed on whole oocytes lysatesin order to avoid RNA loss during its isolation. Amplification ofgenomic DNA was prevented by digesting it with RNase-freeDNase. The method used allowed us specific PCR amplificationand quantification of abundant (e.g. S16 ribosomal protein)and moderate (copper-zinc superoxide dismutase) mRNAs fromas few as two oocytes, while specific amplification andquantification of less abundant transcripts (Metaxin) required 8oocytes as starting material. PCR amplification using thistechnology provides a unique opportunity to study geneexpression in conditions where the amount of experimentalmaterial is very limited.


REFERENCES

  1. Foley KP, Leonard MW, Engel JD. Quantitation of RNA using the polymerasechain reaction. Trends Genet 1993; 9(11): 380-5.

  2. Morrison TB, Weis JJ, Wittwer CT. Quantification of low-copy transcriptsby continuous SYBR Green I monitoring during amplification. BioTechniques1998; 24(6): 954-962.

  3. López-Alańón D M, del Mazo J. Cloning and characterization of genesexpressed during gametogenesis of female and male mice. J Reprod Fertil1995; 103(2): 323-9.

  4. López-Fernández LA, del Mazo J. Characterization of genes expressedearly in mouse spermatogenesis, isolated from a subtractive cDNA library.Mamm. Genome 1996; 7(9): 698-700.

  5. Steuerwald N, Cohen J, Herrera RJ, Brenner CA. Quantification of RNA insingle oocytes and embryos by real-time rapid cycle fluorescence monitoredRT-PCR. Mol Hum Reprod 2000.

  6. (5): 448-53.6.De Felici M, Dolci S. Cellular interactions of mouse fetal germ cells in invitro systems. Curr Top Dev Biol 1987; 23:147-62.

  7. Dulac C. Cloning of genes from single neurons. Curr Top Dev Biol 1998;36: 345-59.

  8. Bustin, S.A. Absolute quantification of RNAm using real-time reversetranscription polymerase chain reaction assays. J Mol Enocrinol 2000;25(2): 169-93

  9. Simone NL, Bonner RF, Gillespie JW, Emmert-Buck MR, Liotta LA. Laser-capture microdissection: opening the microscopic frontier to molecularanalysis. Trends Genet 1998; 14(7): 272-6.

  10. Aslanidis C, Nauck M, Schmitz G. High speed prothrombin G-A 20210 andmethylene- tetrahydrofolate reductase C-T 677 mutation detection usingreal-time fluorescence PCR and melting curves. BioTechniques 1999;27(2): 234-8.




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