PCR sample in pathology
There are three PCR sample in pathology:
1.Fresh tissue
Rearrangement of the BCL-2 gene is the molecular consequence of the t(14;18) chromosomal translocation, which is found in approximately 60-90% of follicular lymphomas. To investigate the ability of the polymerase chain reaction (PCR) to detect this rearrangement in fixed-tissue samples, we studied 48 cases of follicular lymphoma using DNA extracted from paired samples of fresh-frozen tissue and formalin-fixed, paraffin-embedded tissue. A standard phenol-chloroform DNA extraction method was used for both types of tissue. Rearrangements of the major breakpoint region (MBR) and minor cluster sequence (MCS) were examined. Three segments of the human beta-globin gene were also amplified to estimate the degree of DNA degradation in the fixed-tissue samples. PCR of fresh-tissue (intact) DNA revealed amplifiable products in 29 of the 48 follicular lymphomas (60%), whereas the fixed-tissue (degraded) DNA studies were positive in 24 (50%). MBR products were detected in 24 fresh-tissue samples, and varied from 80 bp to > 1.5 kb. Twenty of these cases yielded MBR products in the corresponding fixed-tissue DNA, ranging from 80 to 276 bp. Five fresh-tissue and four fixed-tissue samples produced MCS segments that ranged from 340 bp to 1.2 kb. Four of the five samples with no detectable MBR or MCS translocations using degraded DNA had products greater than 1.0 kb in the fresh-tissue studies. A 175-bp segment of the beta-globin gene was amplified in all 29 fixed-tissue samples; a 324 bp fragment was produced in 20 samples (69%), and a 676 bp segment was detected in 13 (45%) (Liu, 1993).
2. Paraffin block
One of the most active research areas in molecular pathology is retrospective studies on archival tissue samples. However, isolating high-quality genomic DNA from formalin-fixed, paraffin-embedded tissue can be difficult because only minimal amounts of intact DNA may be present in the sample.1 Because of this, analysis of the recovered DNA is generally limited to PCR, and amplification of small target sequences (300 bp or less) is most successful.
The MasterPure™ Complete DNA and RNA Purification Kit was designed to isolate DNA and RNA from a variety of sources, including samples containing small amounts of nucleic acid. Here, we provide a protocol for isolating PCR-ready DNA from paraffin-embedded tissue using the MasterPure Complete Kit. We isolate genomic DNA from a biopsy specimen and show that the DNA is suitable as a template for PCR by amplifying a region of the Factor V gene.
Methods and Results
DNA isolation from breast cancer tissue paraffin sections
The protocol for treatment of formalin-fixed, paraffin-embedded tissue samples prior to purification using the MasterPure Complete DNA and RNA Purification Kit is summarized in Table 1. The isolation of genomic DNA from a breast cancer tissue section was performed following these guidelines. Specifically, DNA was isolated from 0.02 g of 35 µm thick paraffin-embedded samples. (Thin paraffin sections allow the best recovery and quickest extraction times). Five milliliters of xylene were added to the tissue and the sample was incubated for 10 minutes to extract the paraffin. The xylene was poured off and the extraction was repeated. Five milliliters of 100% ethanol were then added and the sample was incubated for 10 minutes. The ethanol was decanted and the ethanol extraction was repeated. The last traces of ethanol were removed by aspiration and the tissue was resuspended in 300 µl of Tissue and Cell Lysis Buffer 1 containing 1 µl of 50 mg/ml Proteinase K. The sample was incubated at 37°C for 30 minutes. The sample was then treated with 150 µl of Protein Precipitation Reagent, mixed by vortexing, and centrifuged for 10 minutes in a microcentrifuge. The DNA-containing supernatant was transferred to a clean microcentrifuge tube and 500 µl of isopropanol were added. The tube was inverted 30 times and then centrifuged for 10 minutes at 4°C in a microcentrifuge. The nucleic acid pellet was then washed twice with 70% ethanol and resuspended in 50 µl of TE buffer.
Table 1. Protocol for the Extraction of DNA from Paraffin-Embedded Tissue.
1. Weigh out 0.01-0.05 g of a 35 µm thick paraffin section.
2. Add 1-5 ml of xylene or Hemo-D (Fisher Scientific) to the paraffin section and incubate for 10 minutes at room temperature to extract the paraffin. Pour off the xylene or Hemo-D.
3. Repeat step 2.
4. Add 1-5 ml of 100% ethanol and incubate for 10 minutes. Pour off the ethanol.
5. Repeat step 4.
6. Remove the last traces of ethanol by aspiration.
7. Continue with the MasterPure Complete protocol2 for nucleic acid recovery. (Note: the standard protocol requires only a 15-minute incubation of the sample with Tissue and Cell Lysis Buffer, whereas a 30-minute to 18-hour incubation is required for paraffin-embedded tissue samples (Masterpure).
Factor V amplification using the isolated DNA
One microliter of the human genomic DNA sample (2% of the total isolated) was used to amplify a 267 bp region of the Factor V gene. The sequences of the primers used were: 5'-TGTTATCACTGGTGCTAA-3' and 5'-TGCCCAAGTGCTTAACAAGACCA-3'. The 50 µl reaction contained 1X MasterAmp™ PCR Optimization Kit PreMix B (1X PCR buffer, 2.5 mM MgCl2, 200 µM each dNTP) (Epicentre), 50 pmoles of each Factor V primer, 1.25 units of MasterAmp AmpliTherm™ DNA Polymerase (Epicentre), and 1 µl of the genomic DNA template. Forty cycles of amplification were performed with the following profile: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds. Five microliters of the sample were separated on a 2% agarose gel and the gel was stained with ethidium bromide. Figure 1 shows that the 267 bp fragment was easily amplified (Shimizu, 1995).
3.Microdissected tissue section
Tissue sections need to be reviewed and annotated prior to microdissection to histologically identify the desired cells for microdissection. Therefore, an evaluation of the tissue samples by a pathologist or a scientist trained in histologic cell identification of frozen tissues is needed before, during and after microdissection. Also of importance is the orientation of the specimen in the tissue block. This is particularly important so that the cells of interest are adequately represented on the slide. Pathology slide review includes the evaluation of the tissue integrity, histopathology, determination of the adequacy of the sample for microdissection based on the amount of the target cell population, and annotation of the target cells on the slide. The pathologist also can give advice on the staining procedure that will help to better identify the cells of interest under the microscope during dissection.
· The goals of histopathology consideration before proceeding with tissue microdissection are: 1) to evaluate the total amount of tissue and the amount of the target cells in the tissue section present in the block, 2) to study the histopathology of the tissue specimen and identify the target cells, and 3) to plan the microdissection for each specimen, taking in account the heterogeneity of tissue samples.
· Always make a regular H&E slide for histopathologic analysis before proceeding with tissue microdissection. Traditional H&E staining (using longer times in each solution than H&E for LCM) and cover slipping of sections 1, 5, and 10 for histopathologic analysis are recommended prior to beginning the study. These sections will serve as a permanent record of tissue specimen status and show histologic changes that occur in the deeper sections.
· Always label the slides with the tissue block identification label, the number of the recut (e.g. 1, 2, 3, etc.), and the date the section was cut from the tissue block.
· If you are unsure of the tissue histology represented in the traditional H&Es, consult with a pathologist to review the slides to accomplish the general goals listed above.
· Keep the traditional H&Es used for histopathologic assessment with you when performing the tissue microdissection. These slides will help in identifying the cells of interest in the tissue section being used for microdissection. Both slides can be compared side by side.
· Only dissect cells that can be clearly identified. If there is any doubt, do not dissect it without consulting with a pathologist (Heidi, 2009).
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