1. A critical decision in designing experiments to interrogate allele-specific gene expression relates to the choice of cell or tissue type for analysis. Allele-specific effects may be highly context dependent, relating not only to cell and tissue type, but also to the nature and kinetics of cell stimulation, and whether cells are of human or animal origin. Pragmatic considerations of the cell numbers required for analysis may limit investigation of primary cells; for approaches such as ChIP, LCLs provide an attractive model.

2. Genotype may also be derived by direct DNA sequencing, restriction enzyme digestion, primer extension, or any other genotyping method of choice.

3. For the approaches described here, the genetic markers chosen should be SNPs. The choice of marker SNP may be derived from databases of publically available SNPs or by de novo analysis through, for example, resequencing. The National Center for Biotechnology Information maintains a public SNP database, dbSNP, available at Other resources for SNP analysis and discovery include For transcript analysis, transcribed exonic SNPs are used, although there are reports of using intronic SNPs to analyze heteronuclear RNA (19). For haploChIP, any SNP within 1 kb 5' or 3' to a gene can be used for analysis of Pol II loading. There is an advantage to identifying the haplotypic structure of the gene region of interest at the outset both to interpret observed allelic differences and to have additional SNP markers with which to confirm any observed allelic differences. Many studies have shown that for a given marker SNP, only a minority of individuals show any allelic difference. This highlights both the need to analyze a sufficiently large number of individuals to have statistical power to detect an effect and the importance of understanding the underlying haplotypic structure, which may otherwise confound effects.

4. Additional discrimination can be achieved by incorporating further mismatches into the primer, for example, changing the third or fifth base from the 3' end. In general 20-bp primers are used with a Tm around 60°C.

5. The protocols below describe the analysis of LCLs, but the principles can be applied to cell or tissue types.

6. The experimental design may be modified to only include unstimulated cells looking at constitutive expression or may span a range of time points following stimulation with, for example, mitogen (ionomycin 125 nM + PMA 200 nM).

7. A variety of different commercial kits are available for isolation of RNA.

8. Fresh formaldehyde should be used from an unopened stock, taking appropriate safety precautions for use and disposal. For a given cell type, the time and temperature of exposure to crosslinking with formaldehyde may need to be titrated.

9. The use of 10% fetal bovine serum in the final PBS wash appears to help protect chromatin integrity in freeze-thawing.

10. Many alternative protocols are available for RNA extraction including those based on spin cup technologies that bind RNA in the presence of a chaotropic salt such as the Absoloutely RNA miniprep kit (Stratagene); these allow for DNase I treatment on the spin cup and avoid the need for phenol-chloroform precipitations steps.

11. A number of different reverse transcriptases are available, and some investigators favor oligo(dT) primers for cDNA synthesis.

12. ChIP lysis buffers 1 and 3 should be chilled and kept on ice; 1X complete protease inhibitor, benzamidine, TLCK, TPCK, and pepstatin should be added to buffers immediately before use. For analysis of phosphorylated Pol II, 10 mM (final concentration) sodium pyrophosphate (Sigma) should be used in all ChIP buffers including nuclear isolation buffers and those used subsequently for immunoprecipitation.

13. Effective sonication can be achieved with a 3-mm microtip on a Branson 450 Soni-fier using constant power with a 30-s constant burst and allowing the suspension to cool on ice for 1 min between pulses. To minimize foaming during sonication, place the probe at mid-depth in suspension and then turn on the power; start at lower settings and then increase (for example, starting at setting 4 (six times) then increasing to 5 (six times)). Sonication should result in DNA fragments approximately 500 bp to 3 kb in apparent size when assessed by gel electrophoresis at this stage (the actual size will be smaller). To assess this, run 10 to 12 ^L of sonicated material on a 1.4% TAE agarose gel. To avoid samples aggregating in the wells of the gel, add Sarkosyl to 0.5% (final concentration) to samples prior to loading on the gel. If the average size of chromatin fragments is greater than several kb, repeat sonication.

14. For some chromatin immunoprecipitation experiments, it may be useful to purify chromatin by cesium chloride ultracentrifugation following sonication. The need for this appears to be dependent on the protein to be immunoprecipitated and the quality of the antibody used for immunoprecipitation. However, the majority of investigators no longer include the cesium chloride centrifugation step.

15. The wash steps are critical to the success of the immunoprecipitation and should be done as carefully as possible to minimize nonspecific background. Experimental conditions may need to be titrated for the individual protein of interest (for example, the stringency of salt and SDS concentration).

16. The choice of method for allelic quantification will be influenced by factors such as cost, availability of technology, and throughput requirements. For all approaches it is essential that appropriate positive and negative controls be included. To assess the accuracy of the technique, the two allelic species can be prepared and mixed in different proportions so that the observed and actual allelic ratios can be compared. Analysis of genomic DNA heterozygous for the marker polymorphism provides a useful baseline for the expected 1:1 allelic ratio, although this may be influenced by other factors such as epigenetic modifications.

17. Primer extension with detection by MALDI-TOF MS is a highly sensitive quantitative approach. To minimize variance, replication should be included at different levels: these could include use of independent LCLs of a given genotype, multiple replicate immunoprecipitation reactions (typically three), replicate PCR amplification of immunoprecipitated DNA fragments (typically four), replicate spotting of PCR products on detection chip (typically four), and independent reads by the mass spectrometer of the spotted PCR amplified material (typically five).

18. An alternative approach is to use TaqMan probes for quantification (3,14).

19. Real-time quantitative ARMS quantitative PCR has also been reported (20).


The author's work is supported by a Wellcome Trust Senior Research Fellowship in Clinical Science.


1. Yan, H., Yuan, W., Velculescu, V. E., Vogelstein, B., and Kinzler, K. W. (2002) Allelic variation in human gene expression. Science 297, 1143.

2. Cowles, C. R., Hirschhorn, J. N., Altshuler, D., and Lander, E. S. (2002) Detection of regulatory variation in mouse genes. Nat. Genet. 32, 432-437.

3. Lo, H. S., Wang, Z., Hu, Y., et al. (2003) Allelic variation in gene expression is common in the human genome. Genome Res. 13, 1855-1862.

4. Morley, M., Molony, C. M., Weber, T. M., et al. (2004) Genetic analysis of genome-wide variation in human gene expression. Nature 430, 743-747.

5. Buckland, P. R. (2004) Allele-specific gene expression differences in humans. Hum. Mol. Genet. 13 Spec No 2, R255-260.

6. Pastinen, T. and Hudson, T. J. (2004) Cis-acting regulatory variation in the human genome. Science 306, 647-650.

7. Knight, J. C. (2004) Allele-specific gene expression uncovered. Trends Genet. 20, 113-116.

8. Hudson, T. J. (2003) Wanted: regulatory SNPs. Nat. Genet. 33, 439-440.

9. Peltonen, L. and McKusick, V. A. (2001) Genomics and medicine. Dissecting human disease in the postgenomic era. Science 291, 1224-1229.

10. Rockman, M. V. and Wray, G. A. (2002) Abundant raw material for cis-regulatory evolution in humans. Mol. Biol. Evol. 19, 1991-2004.

11. Singer-Sam, J., LeBon, J. M., Dai, A., and Riggs, A. D. (1992) A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide. PCR Methods Appl. 1, 160-163.

12. Knight, J. C., Keating, B. J., and Kwiatkowski, D. P. (2004) Allele-specific repression of lymphotoxin-alpha by activated B cell factor-1. Nat. Genet. 36, 394-399.

13. Knight, J. C., Keating, B. J., Rockett, K. A., and Kwiatkowski, D. P. (2003) In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading. Nat. Genet. 33, 469-475.

14. Liu, X., Campbell, M. R., Pittman, G. S., Faulkner, E. C., Watson, M. A., and Bell, D. A. (2005) Expression-based discovery of variation in the human gluta-thione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation. Cancer Res. 65, 99-104.

15. Newton, C. R., Graham, A., Heptinstall, L. E., et al. (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503-2516.

16. Uejima, H., Lee, M. P., Cui, H., and Feinberg, A. P. (2000) Hot-stop PCR: a simple and general assay for linear quantitation of allele ratios. Nat. Genet. 25, 375-376.

17. Butz, J. A., Yan, H., Mikkilineni, V., and Edwards, J. S. (2004) Detection of allelic variations of human gene expression by polymerase colonies. BMC Genet. 5, 3.

18. Takahashi, Y., Rayman, J. B., and Dynlacht, B. D. (2000) Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression. Genes Dev. 14, 804-816.

19. Pastinen, T., Sladek, R., Gurd, S., et al. (2004) A survey of genetic and epigenetic variation affecting human gene expression. Physiol. Genomics 16, 184-193.

20. Bai, R. K. and Wong, L. J. (2004) Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach. Clin. Chem. 50, 996-1001.

0 0

Post a comment