Quantitative Expression of RNA from Frozen Organs and Formaldehyde-fixed and Paraffin-embedded Tissues

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.冻存器官和甲醛固定石蜡包埋组织中RNA的定量表达.目的 定量分析并对比冻存器官和甲醛固定石蜡包埋（formaldehyde-fixed and paraffin-embedded，FFPE）组织中核糖核酸（ribonucleic acid，RNA）的表达情况。 方法 选取不同死亡时间人体脑、心肌和肝组织的冻存及FFPE样本，提取并检测RNA质量浓度，运用实时荧光定量反转录聚合酶链反应（reverse transcription-quantitative polymerase chain reaction，RT-qPCR）技术分析各RNA指标的扩增效率及相对表达量。 结果 与冻存样本相比，FFPE样本中所提取RNA的质量浓度和完整性相对较低。各RNA指标扩增效率与RNA种类及扩增产物长度有关，其中扩增产物较长的甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase，GAPDH）及β-肌动蛋白（β-actin，ACTB）扩增效率不理想，而5S核糖体RNA（5S ribosomal RNA，5S rRNA）扩增效率理想且在各组织中表达稳定，故选为内参指标。较长死亡时间的冻存样本及FFPE样本中扩增产物较长的GAPDH及ACTB表达量下降，而组织特异性微小RNA（microRNA，miRNA）以及扩增产物较短的GAPDH和ACTB在同一组织冻存和FFPE样本中表达具有一致性。 结论 通过标准化RT-qPCR实验，选取合适的RNA指标及设计合适产物长度的引物，能够准确定量FFPE样本中RNA的表达水平。.法医病理学；核糖核酸；石蜡包埋；冷冻保存；反转录聚合酶链反应；核糖核酸完整性指数.