Sivitz W I, Lee E C
Department of Internal Medicine, University of Iowa, Iowa City.
Endocrinology. 1991 May;128(5):2387-94. doi: 10.1210/endo-128-5-2387.
We used a novel adaptation of the polymerase chain reaction to examine relative levels of mRNA encoding two members of the facilitative glucose transporter gene family, the GLUT1 or erythrocyte/HepG2/brain isoform and the GLUT4 or insulin-regulatable isoform. The method was fast (vs. hybridization methods), required no specific probe, and used total RNA samples of less than 1 microgram. Taking advantage of regions of structural similarity and differences between the two isoforms, we designed a single set of oligonucleotide primers capable of amplifying both GLUT1 and GLUT4 cDNAs such that their respective products could be resolved on the basis of a 12 base pair size differential. Hence, reverse transcription and complementary DNA amplification could be carried out for both transcripts using identical primers in the same reaction tube. Using this methodology, we examined the relative amounts of GLUT4 and GLUT1 mRNAs in several rat tissues. As expected based on prior reports using Northern analysis, rat brain contained only GLUT1 mRNA and skeletal muscle contained a large predominance of GLUT4 mRNA. Both isoform mRNAs were found in adipose tissue whereas adipose cells, heart and diaphragm contained predominantly GLUT4 mRNA. Induction of diabetes with streptozocin decreased the GLUT4 to GLUT1 ratio in adipose tissue 4-fold and 24 h of insulin treatment of the diabetic rats increased this ratio 9- to 10-fold. Insulin treatment of normal rats increased this ratio by 70%. Hindlimb skeletal muscle GLUT4 mRNA was quantified in diabetic and insulin-treated diabetic rats as a function of brain GLUT1 mRNA added as an internal standard. Using this methodology, no significant difference in muscle GLUT4 mRNA was noted as a result of 24 h of insulin therapy. In summary, quantitative PCR may be used to compare mRNA levels encoding specific members of a gene family either within given cells or tissues or as affected by physiological perturbations. Subject to certain limitations discussed within, this methodology may be useful in future measurements of glucose transporter mRNA, especially when only small tissue or cell samples are available.
我们采用聚合酶链反应的一种新改良方法，来检测易化性葡萄糖转运蛋白基因家族两个成员编码的mRNA的相对水平，这两个成员分别是GLUT1（红细胞/肝癌细胞系HepG2/脑异构体）和GLUT4（胰岛素可调节异构体）。该方法速度快（与杂交方法相比），无需特异性探针，且使用的总RNA样本量少于1微克。利用这两种异构体之间结构相似和不同的区域，我们设计了一套寡核苷酸引物，能够扩增GLUT1和GLUT4的cDNA，使它们各自的产物能够基于12个碱基对的大小差异得以区分。因此，可在同一反应管中使用相同引物对两种转录本进行逆转录和互补DNA扩增。利用这种方法，我们检测了几种大鼠组织中GLUT4和GLUT1 mRNA的相对含量。正如先前使用Northern分析的报道所预期的那样，大鼠脑仅含有GLUT1 mRNA，而骨骼肌中GLUT4 mRNA占主导地位。脂肪组织中发现了两种异构体的mRNA，而脂肪细胞、心脏和膈肌主要含有GLUT4 mRNA。用链脲佐菌素诱导糖尿病使脂肪组织中GLUT4与GLUT1的比例降低了4倍，而对糖尿病大鼠进行24小时胰岛素治疗使该比例增加了9至10倍。对正常大鼠进行胰岛素治疗使该比例增加了70%。以添加的脑GLUT1 mRNA作为内标，对糖尿病大鼠和胰岛素治疗的糖尿病大鼠后肢骨骼肌中的GLUT4 mRNA进行了定量分析。使用这种方法，胰岛素治疗24小时后，未发现肌肉GLUT4 mRNA有显著差异。总之，定量PCR可用于比较给定细胞或组织内或受生理扰动影响的基因家族特定成员编码的mRNA水平。尽管存在文中讨论的某些局限性，但这种方法可能在未来葡萄糖转运蛋白mRNA的测量中有用，特别是当只有少量组织或细胞样本可用时。
