Use of BIOMED-2 protocols with DNA extracted from paraffin-embedded tissue biopsies and development of control gene primer set（2003）

Background

The integrity of DNA extracted from paraffin-embedded samples and its amplification by PCR are affected by a number of factors such as thickness of tissue, fixative type, fixative time, length of storage before analysis, DNA extraction procedures, and the coextraction of PCR inhibitors.
The integrity of DNA fragments extracted from paraffin-embedded samples also depends on the length of time the blocks have been stored with the best results usually obtained from blocks less than 2 years old, while blocks over 15 years old tend to yield very degraded fragments.

Primer design

Initially, five pairs of control gene PCR primers were designed to amplify products of exactly 100, 200, 400, 600, and 1000 bp in order to assess the quality of DNA submitted for analysis in the BIOMED-2 program. The following target genes were selected:** human thromboxane synthase gene** (TBXAS1, exon 9; GenBank accession no. D34621), human recombination activating gene (RAG1, exon 2; GenBank accession no. M29474), human promyelocytic leukemia zinc-finger gene(PLZF, exon 1; GenBank accession no. AF060568), and human AF4 gene (exon 3; GenBank accession no. Z83679, and exon 11; GenBank accession no. Z83687)

Results of initial testing phase

The primer pairs were tested in separate reactions and subsequently in multiplex reactions using high molecular weight DNA. Owing to the large size range of the products (100–1000 bp), it was necessary to vary the ratio of primer concentrations to obtain bands of equal intensities in the multiplex reactions. However, it proved extremely difficult to be able to amplify all the bands reproducibly and it was decided that the 1000 bp product was probably unnecessary. By increasing the MgCl2 concentration to 2 mM and adding the primers in a 1:1:1:2 ratio, it was possible to reproducibly amplify four bands (100, 200, 400, and 600 bp) of equal intensity from high molecular weight DNA samples. However, for DNA extracted from paraffin blocks, it was thought that an extra size marker at 300 bp would be extremely informative and that the 600 bp marker might not be necessary. Using the gene sequence for the 1000 bp marker (PLZF), primers were redesigned to generate a 300 bp product. These were tested successfully both in monoplex reactions and in multiplex reactions combining the 100, 200, 300, 400, and 600 bp primers (see Figure 13a).

Thus, two primer sets are available for assessing the quality of DNA for amplification:

1. The 100, 200, 300, and 400 bp primers used at 2.5 pmol each can be used for assessing DNA from paraffin-embedded tissues.
2. The addition of the 600 bp primers at 5 pmol allows this set to be used to check the quality of any DNA sample for use with the BIOMED-2 primers and protocols.

Results of general testing phase

In all, 45 paraffin-embedded biopsies were collected corresponding to 30 of the B-cell malignancies, eight of the T-cell malignancies, and seven of the reactive lymphoproliferations submitted as fresh/frozen tissue samples for Work Package 1. The age of the paraffin blocks as well as the methods of fixation and embedding of the samples varied between National Networks.Five sections (10 um each) were cut from the paraffin blocks and DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN) following the manufacturer’s protocol for isolation of genomic DNA from paraffin-embedded tissue.

DNA sample concentration and integrity were estimated by spectrophotometry and by comparison of sample DNA with known standards on agarose-gel electrophoresis.

DNA samples(100 ng) were then analyzed for integrity and amplifiability using the control gene PCR primers (100–400 bp). In the control gene PCR reaction of 24/45 cases, the amplified products were at least 300 bp, whereas in the remaining 21 samples the amplified products were 200 bp or less. No clear correlation between the quality of the DNA and the age of the block or fixation method could be demonstrated. Therefore, it is likely that a combination of factors is responsible for the DNA quality in these samples.

PCR inhibitors are known to be present in DNA extracted from paraffin samples. Dilution of the DNA sample may reduce the concentration of these inhibitors to levels that allow successful amplification to occur. The results in Table 23 show that dilution of the DNA samples has a significant effect on size of the PCR products in the control gene PCR. Overall, 24/45 cases (53%) showed an increased efficiency of amplification when diluted from 100 to 50 ng. The optimal DNA concentration appears to be between 50 and 100 ng, whereas the use of 500 ng appears to inhibit the amplification of large products (300 bp or above). More importantly, 5 ng of DNA has no advantage over a dilution to 50 ng of DNA.

Conclusion

In conclusion, the BIOMED-2 protocols work well with DNA extracted from paraffin-embedded material provided that the DNA can amplify products of 300 bp or more in the control gene PCR.

参考资料：

1. van Dongen, J., Langerak, A., Brüggemann, M. et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17, 2257–2317 (2003).