一种改进的异硫氰酸胍法结合硅胶膜离心吸附柱提取孕妇外周血微量胎儿RNA的方法

孕妇外周血中存在胎儿RNA为无创性产前诊断提供了基础.但血液中富含RNA酶和微量胎儿RNA的特点，对从孕妇血浆中提取胎儿RNA带来困难. 我们以ε血红蛋白基因和胎盘特异表达基因4（PLAC4）mRNA作为研究对象，用改进的异硫氰酸胍法结合硅胶膜离心吸附柱法探索孕妇外周血中胎儿微量RNA的提取方法，获得满意效果. 30例孕妇和9例非孕妇外周血样品中总RNA经凝胶电泳测定显示3条带，分别为28S, 18S和 5.8S. 其28S条带亮度为18S亮度的2倍.总RNA质量浓度（A260／A280）为1.97 g/L，光密度比值(A260-A320）/（A280- A 320）为1.86. 30例孕妇外周血样本有7例提取到ε血红蛋白基因mRNA，ε血红蛋白基因 mRNA 的最小浓度为0.537 μg/mL，最大浓度为1.79 μg/mL，ε血红蛋白基因mRNA的浓度中位数为124 μg/mL. 30例孕妇外周血样本提取到PLAC4 基因mRNA，浓度最小值为2.105×103 copies/mL，最大值为12.760×103 copies/mL，而9例非孕妇中均未提取到（P＜0.01），浓度中位数为6.612×103 copies/mL. 因此,改进的异硫氰酸胍法与硅胶膜离心吸附柱纯化法相结合，可有效抑制RNA降解，用于提取、纯化孕妇血液中微量胎儿RNA.

Extraction of Trace Fetal RNA from Peripheral Blood of Pregnant Women by Improved Metuod of Guanidinium Isothiocyanate Combination with Absorptive Spin Column

Recently,a number of investigators have shown that in addition to DNA,circulating fetal RNA is also present in the maternal plasma.Its presence has opened up new possibilities for noninvasive prenatal diagnosis.But it is difficult to extract fetal RNA from plasma of pregnant women because of abundant RNase and trace fetal RNA.An improved method were used for extracting total RNA from whole peripheral blood of pregnant women.Thirty blood samples from normal pregnancies and nine samples from non-pregnant women were studded.The blood samples were collected in EDTA-containing RNase free plain tubes.For plasma samples,the improved method of guanidinium isothiocyanate combined with spin column and the reagent kit method were applied to extract total RNA.Then RT-PCR was used for the target genes mRNA,ε hemoglobin gene and PLAC4(placenta-specific 4) gene.The extracted total RNA showed three bright bands in agarose gel electrophoresis,28S,18S and 5.8S.The band of 28S was twice than that of 18S.Ratio of A260/A280was 1.97;(A260-A320)/(A 280-A 320)was 1.86.Seven positive and 23 negative ε hemoglobin gene mRNA were obtained from 30 samples.The minimum concentration of ε hemoglobin gene mRNA was 0.537 μg/mL,the maximum concentration was 1.79 μg/mL,the median was 1.24 μg/mL.There was a negative correlation between the amount of ε hemoglobin gene mRNA and gestation age durine 7~10 weeks;Also,PLAC4 mRNA is present in peripheral blood of pregnant women but not in 9 cases of non-pregnant women(P<0.01).The minimum concentration of PLAC4 mRNA was 2.105×103 copies/mL,the maximum concentration was 12.760×103 copies/mL,the average was 6.612×103 copies/mL.The results proposed that improved guanidinium isothiocyanate combined with spin column method is a swift and efficient method for extracting total RNA from whole blood.The potential achievement of these goals would allow us to make a significant step toward the development of a universal marker for noninvasive prenatal gene expression profiling of the fetus by maternal plasma analysis.