1. The in situ RT-PCR method
Our ability to analyze gene function is surpassed by the advances in high throughput DNA sequencing and bioinformatic gene discovery, requiring development of more efficient means to examine expression at the cellular level. To redress that, we developed a PCR-based method to detect mRNA species in situ, in which all of the steps following fixing and sectioning are carried out in liquid phase in a 96-well microtiter tray and only the final step of signal detection is performed on a microscope slide. Being amenable for robotic processing, the in-well in situ RT-PCR method permits high throughput studies of cellular localization of gene transcripts and may become a powerful tool for functional genomics studies (Koltai and Bird 2000, Plant Physiol. 123, 1203-1212). Briefly, plant tissues were fixed in FAA (63% ethanol, 5% acetic acid, 2% formalin) and embedded in low melting point agarose. Sections (40-70 µm) were cut with a Vibratome microtome and individually transferred to a well of a thin-wall polycarbonate 96 well plate containing 700 U mL-1 RNase inhibitor in 45 µL sterile water. The sections were subjected to reverse transcription followed by 30 to 40 PCR cycles using gene-specific primers and digoxigenin (DIG) -11-dUTP. Following the PCR supernatant from wells was aspirated and transferred to a new tube. For the no primer and no Taq controls the PCR was performed with no primers or no Taq, respectively. Following washing and blocking, anti-DIG, alkaline phosphatase conjugated monoclonal antibody was added to the microtiter wells to detect the DIG-labeled PCR products. Sections were then gently removed from each well and stretched on microscope slides in a 50 µL drop of 300 µg mL-1 NBT and 183 µg mL-1 BCIP. Development of the purple color was monitored by microscopy and stopped by rinsing sections with ultra pure water. Sections were observed and photographed on Kodachrome 64T film using a Zeiss Axiophot microscope equipped with Nomarski Differential Interference Contrast optics.
