Gray-Mitsumune M, Abe H, Takahashi J, Sundberg B, Mellerowicz E J
Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå 901 83, Sweden.
Plant Biol (Stuttg). 2004 Jan-Feb;6(1):47-54. doi: 10.1055/s-2003-44747.
We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.
我们探索了一种用于木本茎组织基因表达研究的快速原位逆转录聚合酶链反应（RT-PCR）方案。使用荧光染料偶联核酸进行原位RT-PCR，并使用共聚焦激光扫描显微镜检测来自靶RNA的荧光信号。通过进行两轮PCR反应极大地提高了信号背景比，第一轮不使用荧光染料，第二轮使用染料。使用该方案，我们在次生茎组织中获得了强的基因特异性信号。这些信号依赖于PCR，如在PCR反应中省略DNA聚合酶或引物的组织切片中缺乏细胞质信号所示；并且依赖于RNA，如在RT反应前用核糖核酸酶处理切片时细胞质信号大幅减少所示。为了验证我们的方案，在杂交白杨（Populus tremula L. x tremuloides Michx.）的次生茎中检测了rbcS基因的转录本定位，并与叶绿素自发荧光信号进行了比较。来自rbcS基因的原位RT-PCR信号和叶绿素自发荧光在相同细胞类型中共定位。该信号也通过对从形成层和发育中的木质部分离的RNA进行Northern印迹分析得到证实，从而证实了该方案的有效性。讨论了原位转录本定位的一些困难以及次生组织中信号分布的解释。
