Zbigniew Przybecki, Ewa Siedlecka, Marcin Filipecki, and Ewa Urbanczyk-Wochniak
In situ detection techniques allow specific nucleic acid sequences to be exposed in morphologically preserved tissue sections. In combination with immunocytochemistry, in situ detection can relate microscopic topological information to gene activity at the transcript or protein levels in specific tissues. The advantage of in situ methods over the conventional techniques (e.g., Northern blot, reverse transcription polymerase chain reaction [RT-PCR], or real-time PCR) is that they allow the investigatation of the putative spatial distribution of nucleic acid products activity in a heterogeneous cell population. In this chapter, we describe a protocol for in situ RT-PCR detection of specific messenger RNA in cucumber (Cucumis sativus), although this protocol can be used for any plant species, floral buds, and somatic embryo tissue sections on glass microscope slides. A successful in situ RT-PCR procedure requires the optimization of many conditions related to the tissue types used, for example, a cell's age, size, and composition, which may influence the detection of RT-PCR products, as well as specific transcript availability. Moreover, parameters, such as the fixation time, thermal cycling set-up, and the time of detection of RT-PCR products, also should be optimized. The importance of the other factors also is estimated in the protocol. In addition several types of controls that are necessary for a trustworthy in situ RT-PCR method are being discussed.
Key Words: Direct in situ RT-PCR; Cucumis sativus, floral buds; somatic embryos; paraffin tissue sections; digoxigenin-11-dUTP; alkaline phosphatase substrate.
There are several common names for in situ polymerase chain reaction (PCR), which is performed based on RNA. Nuovo (1), who was one of the first developers of the method, called it reverse transcription (RT) in situ PCR, but since then, other names have been created for it, such as in situ complementary (c)DNA(2) or in situ reverse transcriptase PCR (3), which are present in literature to date (4).
From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.
Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ
In situ RT-PCR combines the sensitiveness of PCR amplification with spatial localization of products to monitor the appearance of specific transcripts in the tissue sections. Therefore, in situ RT-PCR defines a powerful tool for the low-abundance transcript detection (5) because the revealing threshold can be as low as one or two copies per cell. In comparison, in situ hybridization detects 10 to 20 copies per cell (6). This method has not been used to a large extent in plants, but it provides several advantages over the classic in situ RNA hybridization, which was widely discussed recently (7).
The first application of in situ RT-PCR for the plant tissue was reported by Woo et al. (8) and described the expression of the HIS 3;2gene (encoding the H1 histone) in the single detached border cells of pea seedlings. The subsequent papers on applying in situ RT-PCR technique to the plant material regarded several different tissues and genes (9-16).
In situ RT-PCR techniques can be classified into two groups based on labeling and detection systems used (Fig. 1). During the direct in situ RT-PCR, digoxigenin (biotin or fluorescein)-labeled nucleotides (13,14) or primers (15) are incorporated into the PCR product, leading to a direct signal detection. To the contrary, the indirect signal detection for in situ RT-PCR occurs when the PCR product is subsequently visualized by hybridization with specifically labeled probe (16). The direct in situ RT-PCR can be a quicker alternative to the indirect technique because it avoids the subsequent in situ hybridization step.
Although RT-PCR technique generally is adjusted by optimizing RT-PCR mixture and timing, for the successful procedure it also is crucial to set up optimal conditions for each new tissue in respect of its cells size and composition (e.g., lignified walls), as discussed previously (7). Engler et al. (17) described an in situ protocol suitable for obtaining the optimal results for different Arabidopsistissues. Lee and Tegeder (7) observed that the thickness of the tissue sections and proteinase K pretreatment strongly increased the probability of successful application of in situ RT-PCR. Furthermore, the fixation and embedding processes also have a noticeable influence on the in situ RT-PCR results (14).
This chapter is aimed at providing a laboratory protocol for the in situ RT-PCR optimized for localization of transcripts in the cucumber floral buds and cucumber somatic embryo tissue sections, which also can be used for other plant species. Additionally, we provide a comprehensive procedure of in situ RT-PCR while discussing the main steps of this technique and providing a detailed list of materials effectively used in our laboratory for it.
DIRECT IN-SITU PCR
Labelled nucleotides or primers in RT-PCR mix reaction labeled nucleotides enzyme linked primary antibody
PLANT TISSUE-SECTION i
ADD RT-PCR REAGENTS
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