Stimulation Protocol

Cell Stimulation Protocol

Initially spin down cells, lyse for RNA extraction, RT-PCR for 20, 25, 30 cycles and do serial dilution of the RNA to avoid saturation (dilue: 50x, 100x, 25x, 500x).

1. For one 90-100% confluent T-75 flask

2. Wash with 10 mL PBS

3. Trypsinize with 1.5 mL trypsin

4. Incubate for 15 minutes at 37 degrees

5.Stop trypsinization by adding 20 mL media to the flask

6. Transfer all to a 50 mL tube

7. Wash flask with 10 mL media

8. Add this 10 mL to the same 50 mL tube

9. Spin 10 minutes at half speed

10. Aspirate media

11. Add 3 mL fresh media

12. Pipet up and down with a 3 mL syringe and a 19.5 gauge needle to break up cell clumps

13. Dilute to 50 mL total volume

14. Plate 2 mL per well in 6-well plates. So one T-75 can plate into 4 X 6-well plates.

15. Allow growth in plate for 24 hours

16. Change media to chracoal stripped media

17. Allow growth in plate for 24 hours

18. If after 24 hr, cells are 80% confluent, change media again to charcoal stripped media 3 mL/well and stimulate with 30 microlitres of steroid and 5′-aza-deoxycytidine.

19. Incubate for 24 hours, harvest 4 and 5 sets for RNA analysis

a. aspirate media

b. wash cells twice carefully with cold PBS

c. trypsinize or scrape

d. add 1 mL of Trizol to each well

e. shake or place on rotor for 15 minutes

f. transfer from each well into a 1.5 mL centrifuge tube

g. proceed with RNA extraction

20. Incubate for 7 days, harvest 1-3 for protein analysis

a. transfer supernatant to 5 mL Falcon tubes

b. spin down cells

c. take supernatant and move on to protein assays and analysis

Total Cellular RNA Extraction Protocol

METHODS and MATERIALS: A Protocol for Total Cellular RNA Extraction

Total cellular RNA is isolated from 60-80% confluent cell monolayers using the RNeasy Midi Kit (QIAGEN Inc., Valencia , CA ) and following the manufacturer’s instructions. Total RNA concentration and its purity can and should be determined spectrophotometrically.

Bisulphite Treatment Methylight

This method is great for detecting minimal amounts of aberrant DNA methylation. Primers are designed that overlap with CpG dinucleotides, and the DNA is treated with sodium bisulphite as mentioned previsouly. The advantage of this method, is that there is no need for any steps after PCR, as it uses fluoresence-based real-time PCR, Taqman, technology.

Southern Blotting Method for DNA Methylation Detection

This method utilized methylation-sensitive restriction enzymes to digest DNA, cleaving at specific sites, which have been altered by DNA methylation, followed by Southern blot analysis of the DNA products. A very simple technique to analyze DNA methylation, however, it is very prone to numerous downfalls: poor sensitivity; very large amounts of DNA are required in order to receive a signal on blotting, and is only to detect methylated CpG’s that are recognized by the enzymes. Overall, there is little control of the sites/outcome of the test by the researcher, and a great deal of work and time must be spent in obtaining large amounts of DNA, and of good quality. Not recommended for any clinical samples/materials, such as biopsies and paraffin-embedded tissues because of this.

J.P.Issa, et al. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat. Genet. (1994) 7: 536-540.

Combined Bisulfite Restriction Analysis COBRA Protocol

COBRA is a great, flexible, and easy method to quantitatively analyze CpG dinucleotides within DNA, at specific gene loci in small amount of DNA. It is a great method to use, as it can quantitatively assess CpG islands. Sodium bisulfite treatment of DNA is the initial step, followed by PCR. Subsequently, restriction digestion of the DNA is carried out. Methylation levels in the original DNA sample are revealed by the differing amounts of digested and undigested PCR products quantitatively. Overall, a great method, especially for paraffin sections, and clinical studies due to its ease and quantitative aspects.

Z. Xiong et al. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res (1997) 24: 5058-5059.

Restriction Endonuclease Enzymes

Restriction Endonuclease Enzymes to Study DNA Methylation and Epigenetic Changes

A few restriction enzymes are known to differential cleave methylated versus non-methylated DNA. These enzymes can be used to help you. When selecting these restriction enzymes, it is crucial to that they leave staggered ends, have a cut site that is different from the recognition site, and cannot be affected through potentially methylated target sequences.

C. Laird et al. Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules. Proc Nat Acad Sci (2004) 101: 204-209.

Chromatin Immunoprecipitation

Chromatin Immunoprecipitation Assay (ChIP)

Chromatin Immunoprecipitation Assay, also known as the ChIP assay allows one to determine the mechanism of trascriptional repression, by immunoprecipitating chromatin with antibodies. ChIP involves formaldehyde treatment of a cell population to cross-link proteins to their target DNA in vivo, cell lysis, sonication to fragment chromatin, and then immunoprecipitation of specific chromatin complexes using an antibody against the protein of interest. After removal of the cross-links, PCR amplification at a selected locus, or slot blot hybridization using a specific probe is performed.

Chromatin Immunoprecipitation Assay / ChIP Advantages

Chromatin Immunoprecipitation Assay / ChIP has the advantage of measuring protein DNA interactions of promoter regions with protein complexes in the natural genomic state. Moreover, the ChIP assay can directly provide information on the histone acetylase status of specific chromatin regions, and be used to study non-histone proteins, even ones that do not directly bind DNA. It also minimizes the chances of chromatin rearrangements during preparation and precipitation, and it can be used with gene-specific primers for increased sensitivity or with a specific probe, slot blot hybridization, for increased specificity.

Chromatin Immunoprecipitation Assay / ChIP Disadvantages

Dificulties with using ChIp will include, the danger that the cross-linking step may fix interactions that are of minor functional sugnificance, precipitation us often very inefficient, and purifying cross-linked DNA-protein usually requires isopycnic centrifugation, a prolonged and expensive procedure. A large number of cells are usually required and the data represents an average of potentially multiple functional states in the cell population. In addition, negative results do not necessarily mean that a given factor is not associated with the site. Certain antibodies are poor in immunoprecipitating crosslinked chromatin and lack of crosslinking can results from inaccessibility of factors in large complexes or biochemical properties of proteins that decrease cross-linking efficiency.

Chromatin Immunoprecipitation Assay / ChIP Success

The likelihood of success with the Chromatin Immunoprecipitation Assay / ChIP method is protein and antibody dependent, but the methods track record suggests that this technique is extremely versatile and widely applicable. Success will depend on the abundance of the desired protein-DNA adducts being studied, the quality and affinity of the antibodies being used, and on the size and complexity of the genetic loci studied.

Alternative Approaches Instead of Chromatin Immunoprecipitation Assay / ChIP

Alternative approaches include, studies using conventional methods such as transient transfection assays, electrophoretic mobility shift assays (EMSA) and coimmunoprecipitation.

ChIP References

Beatrice N et al. Location analysis of DNA-bound proteins at the whole genome level: untangling transcriptional regulatory networks. Bioassays 23, 472-476 (2001)

Weinmann AS et al. Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26, 37-47 (2002)

Johnson KD et al. Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation. Methods 26, 27-36 (2002)

Kuo MH et al. In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19, 425-433 (1999)

Hairpin-Bisulfite Polymerase Chain Reaction PCR

Laird C. et al, published a great paper describing this method that they utilized to assess the fidelity of methylation transmission of CpG islands within the FMR1 gene of human lymphocytes. They generated a hairpin-linker with staggered ends (25 base pairs long), complementary to the targeted cut-site of the DNA, which was used after restriction enzyme digest of the DNA. The linker was attached to the DNA through the use of DNA ligase. The double-stranded target DNA was then subjected to sodium bisulfite treatment, and PCR amplification with designed primers that bound to the linker approximately 200 nucleotides away. The PCR products were then subsequently sequenced.

C. Laird et al. Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules. Proc Nat Acad Sci (2004) 101: 204-209.

Identification of CpG Islands Exhibiting Altered Methylation Patterns (ICEAMP)

This method allows for the identification of large regions of DNA with changes in methylation patterns through the use of methyl-CpG binding domain column which isolates methylated GC-rich sequences from both tumors and surrounding normal tissue. Subtractive hybridization is used subsequently, which removes sequences common to both, thereby leaving only methylated sequences unique to the tumor cells. Such a technique has numerous advantages and is easily one of the best methods to assess CpG islands for clinical studies.

S.Cross et al. Purification of CpG islands using a methylated DNA binding column. Nat. Genet. (1994) 6: 236-244.

Methylation Sensitive Single Nucleotide Primer Extension Ms-SNuPE

This method allows you to analyze several CpG dinucleotides in a single reaction. Paired primer extensions with 32P linked dCTP or TTP will reveal whether a Cytosine is methylated or not. Primers are made that anneal to the PCR template and terminate subsequently 5 prime of the cytosine to be assayed. This will quantitatively assess the ratio of methylated and unmethylated cytosines in the DNA sample at that specific CpG dinucleotide.

Gonzalgo ML et al. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res (1997) 25: 2529-2531