乳腺癌冰冻和石蜡包埋样本的RNA以及基因表达情况的比较研究

Analysis of RNA and gene expression from frozen and paraffin-embedded breast cancer tissues

目的比较乳腺癌新鲜冰冻和石蜡包埋样本中提取总RNA的质量，并分析其基因表达情况。方法收集新鲜冰冻及其石蜡包埋的人乳腺癌组织共5对，另取10例保存时间为1-10年不等的人乳腺癌组织石蜡样本。试剂盒提取总RNA，用随机引物实时定量聚合酶链反应（qRT—PCR）分析新鲜冰冻与其石蜡样本的mRNA表达情况，并对不同保存时间的石蜡样本比较扩增管家基因不同产物长度的Ct值差异，以了解RNA降解程度。结果新鲜冰冻后的石蜡样本RNA片段长度弥散分布在200bp左右，扩增90bp长度ACTB基因的有效RNA模板量仅为相应冰冻样本的0．46倍（P〈0．05）。冰冻样本扩增90bp产物的Ct值显著低于其扩增203bp的Ct值（P=0．02），提示冰冻样本RNA有一定降解。石蜡样本mRNA表达与相应的冰冻样本无显著差异（P〉0．05），两种样本目的基因△Ct值经Spearman相关性分析有显著相关性（r=0．954，P=0．000）。扩增不同保存时问石蜡样本的3个长度片段的Ct值均有显著差异，随着扩增片段的增大，Ct值也逐渐增加。结论冰冻样本的RNA相对较完整，质量较好；石蜡样本的RNA有明显降解，但能够准确反映组织mRNA水平表达情况；石蜡样本的Ct值随扩增片段的增大而逐渐增加，能顺利扩增150bp左右长度的产物，为石蜡样本的进一步研究提供实验基础。

Objective To compare the quality of total RNA extracted from frozen and paraffin-embedded breast cancer sampies and their gene expression. Methods We collected 5 paired of frozen and FFPE human breast cancer tissues, and another 10 FFPE samples with storage time from 1 to 10 years. Total RNA was extracted and converted to cDNA with random primers. Gene expressions of two types of samples were detected by real-time fluorescence quantitative PCR reaction. We measured the amplification results of three fragments from ACTB gene to assess the intensity of RNA from FFPE tissues. Results RNA fragments dispersed 200bp in length. Compared to frozen samples, an average 54% loss of intact amplicon template for amplification ACTB （90bp） gene in the corresponding FFPE materials （P 〉 0.05 ）. The integrity of RNA of them had significant differences. The Ct value for amplification of 90bp fragment was lower than that of 203bp fragment （P = 0. 02 ） , which indicated that RNA from frozen samples was relatively intact. No difference in gene expression was found between frozen and FFPE samples （ P 〉 0. 05 ）. And a good correlation of them was derived by Spearman correlation coefficient（r = 0. 954,P = 0. 000）. There were statistical differences between Ct values of three fragments of FFPE tissues. And the Ct value increased with time of the archival samples from which the RNA was extracted, indicating a high level of RNA degradation in these samples. Conclusion RNA from frozen samples is relatively intact. RNA from paraffin samples is obviously degraded, but it can accurately reflect the expression of mRNA. Ct value gradually increased with the fragment length. 90-150bp is a favorable length of product for amplification. This research provides an experimental basis for further extensive research of FFPE samples.