Optimization Of PCR Conditions For Immunoglobulin Gene Rearrangement In Paraffin-Embedded Tissues Of Non-Hodgkin's Lymphoma

Background and aimsLymphoma is one of the major problematic diseases in clinic pathological diagnosis, which often need to draw support of the other techniques as IHC and gene rearrangement detection. It has always been concerned since McCarthy first used PCR technique to detect antigen receptor gene rearrangement to aid the diagnosis of lymphoma. However, the major obstacle to use technique as a routing is its high false negative rates and inconstant results, especially in paraffin-embedded tissues (PET). To improve these conditions, several aspects have been investigated, including primer analysis and design for Immunoglobulin (Ig) gene rearrangements, optimizing conditions for these primers, optimizing of the DNA extraction method from PET, and comparison their detecting rates among different primers in PET lymphoma tissue.Methods1. More than fifteen pairs of primers, which have ever been used for detecting Ig gene rearrangement, were analyzed and compared with their germline gene fragments by using Clustal/W software. All these primers including newly designed primers were testified and analyzed by PrimerPremier 5.0 software.2. The PCR conditions of all these primers were optimized by means of assessing gene rearrangements of DG75 cell lines (for B cells) and Jurkat cell lines (for T cells). Almost all these primers PCR products were sequenced, and were analyzed by Clustal/W and Blast software on the web.3. By using 21 cases of lymphoid PET tissues, three DNA extraction methods, including simply digestive tissue, Phenol/chloroform extraction and Commercial kit (QIAamp DNA minikit), were evaluated by the measure of the concentration, the OD2&0/OD280 ration of extracted DNA and the results of PCR.4. Eight pairs of gene rearrangement primers, seven for Ig and one for TCRy, were used to detect the gene rearrangements of 144 blocks of lymphoid PET tissues.Results1. Four new pairs of primers, two for IgH chain and two for IgL chain, were designed after having contrasted fifteen pairs of Ig gene rearrangement primers. All Fourth pairs of newly designed primers seemed perfect according to the result of Primer designer Software.2. The conditions of eight pairs of primers, including four newly designed primers, three pairs of ever used primers and one TCRy primer set, were optimized by assaying the gene rearrangement of DG75 and Jurkat lymphoma cell lines. Three clear bands can be obtained with IgH FR3 primers in the PCR DG75 cell line gene rearrangement, but they are associated with the Ig gene sequence according to the result of Blast. Five sequences from DG75 cell lines have been submitted to GeneBank.3. From the results of DNA OD260/OD280 ratio, Commercial kit method seems to outstand the other two DNA extraction methods. But the results of PCR showed no significant difference by detecting p-globin gene in 21 cases of lymphoid tissues.4. 甌he detection rates of three primer sets P1 (for IgH FR3 region),P2A (for IgH FR2 region) and Pic (for IgH FR1 region) are 71.4%, 62.1% and 36.7% respectively in 49 B cell lymphoma tissues. There was a statistic difference between Pic and P2A or Pic primer sets. (2)The detection rates of primer sets for IgH FR1 region, PI, P2 and P3, are 71.7%, 82.2%, 76.1% respectively in 113 B cell lymphoma detected, and our newly designed primer P2 set get the highest detection rate among three, although there were no statistic differences among them. The detection rate of Ig light chain primers (Pic+P>,) is 71.8% among 39 blocks of B cell lymphoma tissues . (D The best primer pairs, (P1+P2) primer set, were selected after analyzing and comparing several primer combination sets. The detection rate could be raised from 82.3% to 92.9% in B cell lymphoma PET tissue. ㏕he detection rate of TCRy primer was 91.7% among 24 T cell lymphoma of PET tissues.ConclusionAccording to the results of our present research, the following conclusion could be drawn:1. Different immunoglobulin framework region (FR) primer sets showed...