This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.
This method is used to detect genomic DNA deletions in tumor cells. For a more detailed discussion of applying this approach to microdissected samples, see Allelic Loss Studies in Prostate MP at NCI.
3. Time Requirements
TIP: Investigators must be especially careful when using this methodology to analyze archival tissue specimens. Formalin fixation in particular results in DNA that is difficult to amplify and often produces inconsistent PCR results, including artifactual allelic loss and poor amplification of large products. Therefore, when this technique is used to analyze archival samples, it is highly recommended that replicate experiments (multiple independent dissections, triplicate PCR reactions, etc.) be used to verify results.
A: LCM and Proteinase K Treatment
TIP: The number of cells needed to successfully perform the assay varies depending on the quality and processing conditions of the tissue samples. One thousand cells is recommended as a good starting point.
B: Prepare the Glass Plates
TIP: Use Accuwipes for cleaning purposes, as they will not leave lint behind and are non-abrasive.
C: Polymerize the Gel
TIP: Acrylamide is a neurotoxin. Be sure to wear gloves and a labcoat when working with this substance.
- Add 480 µl of 10% ammonium persulfate to 75 ml of Gel-mix-6.
- Mix by inversion.
- Hold the nozzle of the bottle at the corner of the gel cast.
- Hold the gel cast at a 45o angle to the bench and pour the gel between the plates. If bubbles get trapped between the plates, remove them by tapping the outside of the plates or by tipping the plates upright.
- Insert the straight side of the comb approximately 1 cm into the gel. If bubbles are introduced at this point, remove the comb and use the teeth of the comb to sweep out small bubbles.
- Clamp the top of the plates together.
- Allow the gel to polymerize for at least one hour.
TIP: The gel can be left to polymerize overnight. However, if bubbles appear, the gel has begun to separate from the plates.To minimize separation, wrap the gel in plastic film and store at 4°C until use.
D: PCR Reaction
TIP: Investigators must be especially careful when using this methodology to analyze archival tissue specimens. Formalin fixation in particular results in DNA that is difficult to amplify and often produces inconsistent PCR results, including artifactual allelic loss and poor amplification of large products. If this technique is to be utilized for analysis of archival samples, we highly recommend that replicate experiments (multiple independent dissections, triplicate PCR reactions, etc.) be used to verify results.
TIP: DNA that is recovered from microdissected samples and "semi-purified" using a one-step proteinase K buffer will sometimes produce "non-specific" PCR products in addition to the true alleles. Moreover, larger alleles will sometimes amplify much less well than smaller alleles. Thus, normal-cell DNA recovered from the same tissue section as the tumor DNA serves as the best control for determining the presence or absence of allelic loss.
TIP: Be sure to mix the LOH reaction mixture with the DNA sample by pipetting. This is especially critical for DNA from microdissected samples that has been processed through a one-step proteinase K-based "purification."
TIP: Investigators may want to consider the "touchdown" procedure for PCR by Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS: "Touchdown" PCR to circumvent spurious priming during gene amplification. Nucl Acids Res 19:4008, 1991. Advantages include:
- Much cleaner bands, since by starting with a high annealing temperature of 66 degrees and lowering 1 degree every cycle, the first PCR products are the most specific ones.
- The exact same protocol can be used for all primers.
E: Finalize Gel Preparation
F: Gel Loading
TIP: It is best to skip lanes to avoid contamination caused by leaking between the wells.
G: Separate the Gel
TIP: Use MR film for maximum resolution of bands. An intensifying screen is useful when analyzing PCR products from small numbers of microdissected cells.
Debelenko LV, Brambilla E, Agarwal SK, Swalwell JI, Kester MB, Lubensky IA, Zhuang Z, Guru SC, Manickam P, Olufemi SE, Chandrasekharappa SC, Crabtree JS, Kim YS, Heppner C, Burns AL, Spiegel AM, Marx SJ, Liotta LA, Collins FS, Travis WD, Emmert-Buck MR. Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung. Hum Mol Genet 6(13):2285-90, 1997.
Emmert-Buck, MR, Lubensky, IA, Dong, Q, Chandrasekharappa, C, Guru, SC, Manickam, P, Keseter, M, Olufemi, S-E, Agarwal, S, Burns, AL, Spiegel, AM, Collins, FS, Marx, SJ, Zhuang, Z, Liotta, LA, Debelenko, LV. Localization of the multiple endocrine neoplasia Type I (MEN1) gene based on tumor deletion mapping. Cancer Res 57:1855-8, 1997.
Emmert-Buck M R, Vocke C D, Pozzatti R O, Duray P H, Jennings S B, Florence C D, Zhengping Z, Bostwick D G, Liotta L, and Linehan WM. Allelic loss on chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia. Cancer Res 55: 2959-62, 1995.
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