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. 1996 Dec 1;24(23):4812–4816. doi: 10.1093/nar/24.23.4812

A quantitative and specific method for measuring transcript levels of highly homologous genes.

A J Lombardo 1, G B Brown 1
PMCID: PMC146290  PMID: 8972870

Abstract

Because of their high nucleotide sequence homology, the specific detection of mRNA transcripts of individual members of a gene family presents certain problems. Here we apply and defend the single nucleotide primer extension (SNuPE) as a simple, specific, and highly quantitative assay for this purpose. The method distinguishes regions of the brain sodium channel gene family that vary by as little as a single nucleotide. The technique has been modified to include an intersample normalizer, and adaptation of the SNuPE assay to other gene families of interest to neuroscientists should be easy to achieve.

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Selected References

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  1. Bertoni J. M., Brown P., Goldfarb L. G., Rubenstein R., Gajdusek D. C. Familial Creutzfeldt-Jakob disease (codon 200 mutation) with supranuclear palsy. JAMA. 1992 Nov 4;268(17):2413–2415. [PubMed] [Google Scholar]
  2. Brunetto M. R., Randone A., Ranki M., Jalanko A., Piantino P., Giarin M., Capra G., Calvo P. L., Oliveri F., Bonino F. Quantitative analysis of wild-type and HBeAg minus hepatitis B viruses by a sequence-dependent primer extension assay. J Med Virol. 1994 Jul;43(3):310–315. doi: 10.1002/jmv.1890430320. [DOI] [PubMed] [Google Scholar]
  3. Buck K. J., Hahner L., Sikela J., Harris R. A. Chronic ethanol treatment alters brain levels of gamma-aminobutyric acidA receptor subunit mRNAs: relationship to genetic differences in ethanol withdrawal seizure severity. J Neurochem. 1991 Oct;57(4):1452–1455. doi: 10.1111/j.1471-4159.1991.tb08313.x. [DOI] [PubMed] [Google Scholar]
  4. Buzin C. H., Mann J. R., Singer-Sam J. Quantitative RT-PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies. Development. 1994 Dec;120(12):3529–3536. doi: 10.1242/dev.120.12.3529. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Dean A. D., Greenwald J. E. Use of filtered pipet tips to elute DNA from agarose gels. Biotechniques. 1995 Jun;18(6):980–980. [PubMed] [Google Scholar]
  7. Geiger J. R., Melcher T., Koh D. S., Sakmann B., Seeburg P. H., Jonas P., Monyer H. Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron. 1995 Jul;15(1):193–204. doi: 10.1016/0896-6273(95)90076-4. [DOI] [PubMed] [Google Scholar]
  8. Harju L., Weber T., Alexandrova L., Lukin M., Ranki M., Jalanko A. Colorimetric solid-phase minisequencing assay illustrated by detection of alpha 1-antitrypsin Z mutation. Clin Chem. 1993 Nov;39(11 Pt 1):2282–2287. [PubMed] [Google Scholar]
  9. Joho R. H., Moorman J. R., VanDongen A. M., Kirsch G. E., Silberberg H., Schuster G., Brown A. M. Toxin and kinetic profile of rat brain type III sodium channels expressed in Xenopus oocytes. Brain Res Mol Brain Res. 1990 Feb;7(2):105–113. doi: 10.1016/0169-328x(90)90087-t. [DOI] [PubMed] [Google Scholar]
  10. Kayano T., Noda M., Flockerzi V., Takahashi H., Numa S. Primary structure of rat brain sodium channel III deduced from the cDNA sequence. FEBS Lett. 1988 Feb 8;228(1):187–194. doi: 10.1016/0014-5793(88)80614-8. [DOI] [PubMed] [Google Scholar]
  11. Kokaia M., Pratt G. D., Elmér E., Bengzon J., Fritschy J. M., Kokaia Z., Lindvall O., Mohler H. Biphasic differential changes of GABAA receptor subunit mRNA levels in dentate gyrus granule cells following recurrent kindling-induced seizures. Brain Res Mol Brain Res. 1994 Jun;23(4):323–332. doi: 10.1016/0169-328x(94)90242-9. [DOI] [PubMed] [Google Scholar]
  12. Kuppuswamy M. N., Hoffmann J. W., Kasper C. K., Spitzer S. G., Groce S. L., Bajaj S. P. Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1143–1147. doi: 10.1073/pnas.88.4.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lin F. H., Lin R., Wisniewski H. M., Hwang Y. W., Grundke-Iqbal I., Healy-Louie G., Iqbal K. Detection of point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 in Alzheimer's brains. Biochem Biophys Res Commun. 1992 Jan 15;182(1):238–246. doi: 10.1016/s0006-291x(05)80136-6. [DOI] [PubMed] [Google Scholar]
  14. Macdonald R. L., Olsen R. W. GABAA receptor channels. Annu Rev Neurosci. 1994;17:569–602. doi: 10.1146/annurev.ne.17.030194.003033. [DOI] [PubMed] [Google Scholar]
  15. Mandel G., Cooperman S. S., Maue R. A., Goodman R. H., Brehm P. Selective induction of brain type II Na+ channels by nerve growth factor. Proc Natl Acad Sci U S A. 1988 Feb;85(3):924–928. doi: 10.1073/pnas.85.3.924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Noda M., Ikeda T., Kayano T., Suzuki H., Takeshima H., Kurasaki M., Takahashi H., Numa S. Existence of distinct sodium channel messenger RNAs in rat brain. Nature. 1986 Mar 13;320(6058):188–192. doi: 10.1038/320188a0. [DOI] [PubMed] [Google Scholar]
  17. O'Rahilly S., Krook A., Morgan R., Rees A., Flier J. S., Moller D. E. Insulin receptor and insulin-responsive glucose transporter (GLUT 4) mutations and polymorphisms in a Welsh type 2 (non-insulin-dependent) diabetic population. Diabetologia. 1992 May;35(5):486–489. doi: 10.1007/BF02342449. [DOI] [PubMed] [Google Scholar]
  18. Patton D. E., Goldin A. L. A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes. Neuron. 1991 Oct;7(4):637–647. doi: 10.1016/0896-6273(91)90376-b. [DOI] [PubMed] [Google Scholar]
  19. Pellegrini-Giampietro D. E., Pulsinelli W. A., Zukin R. S. NMDA and non-NMDA receptor gene expression following global brain ischemia in rats: effect of NMDA and non-NMDA receptor antagonists. J Neurochem. 1994 Mar;62(3):1067–1073. doi: 10.1046/j.1471-4159.1994.62031067.x. [DOI] [PubMed] [Google Scholar]
  20. Schaller K. L., Krzemien D. M., Yarowsky P. J., Krueger B. K., Caldwell J. H. A novel, abundant sodium channel expressed in neurons and glia. J Neurosci. 1995 May;15(5 Pt 1):3231–3242. doi: 10.1523/JNEUROSCI.15-05-03231.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Singer-Sam J., LeBon J. M., Dai A., Riggs A. D. A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide. PCR Methods Appl. 1992 Feb;1(3):160–163. doi: 10.1101/gr.1.3.160. [DOI] [PubMed] [Google Scholar]
  22. Singer-Sam J. Quantitation of specific transcripts by RT-PCR SNuPE assay. PCR Methods Appl. 1994 Feb;3(4):S48–S50. doi: 10.1101/gr.3.4.s48. [DOI] [PubMed] [Google Scholar]
  23. Singer-Sam J., Riggs A. D. Quantitative analysis of messenger RNA levels: reverse transcription-polymerase chain reaction single nucleotide primer extension assay. Methods Enzymol. 1993;225:344–351. doi: 10.1016/0076-6879(93)25023-u. [DOI] [PubMed] [Google Scholar]
  24. Westbrook G. L. Glutamate receptor update. Curr Opin Neurobiol. 1994 Jun;4(3):337–346. doi: 10.1016/0959-4388(94)90094-9. [DOI] [PubMed] [Google Scholar]
  25. Wong H., Anderson W. D., Cheng T., Riabowol K. T. Monitoring mRNA expression by polymerase chain reaction: the "primer-dropping" method. Anal Biochem. 1994 Dec;223(2):251–258. doi: 10.1006/abio.1994.1581. [DOI] [PubMed] [Google Scholar]
  26. Zhong J., Carrozza D. P., Williams K., Pritchett D. B., Molinoff P. B. Expression of mRNAs encoding subunits of the NMDA receptor in developing rat brain. J Neurochem. 1995 Feb;64(2):531–539. doi: 10.1046/j.1471-4159.1995.64020531.x. [DOI] [PubMed] [Google Scholar]

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