PROTOCOLS


Tissue Preparation

Tissue samples were trimmed to 4 mm or less in thickness and fixed 4 to 24 h (depending on thickness) at 4°C in at least 10 volumes of freshly prepared 3:1 ethanol:acetic acid (Farmer's fixative) according to Ruzin (1999). When needed, tissue in fixative was subjected to 15 min of vacuum to assist sinking and infiltration. Fixed tissue was dehydrated at room temperature in a graded series of ethanol (3 h each [v/v] 75%, 85%, 100%, 100%, and 100%), followed by an ethanol:xylenes series (3 h each [v/v] 75%:25%, 50%:50%, 25%:75% 0%:100%, 0%:100%, and 0%:100%). Flakes of Paraplast-X-Tra tissue embedding medium (Fisher Scientific, Fair Lawn, NJ) were added to the final step. Once the flakes dissolved at room temperature, liquefied Paraplast-X-Tra was added, and sample vials were transferred to an oven at 58°C. The medium was replaced at 3- to 6-h intervals until the odor of xylenes was absent. Samples were positioned in Paraplast-X-Tra, and sections were cut on a rotary microtome (Microtom HM310, Waldorf, Germany), floated in water on Probe-on microscope slides at 42°C to stretch ribbons, air-dried, and stored in darkness at 4°C under dehydrating conditions. For LCM, slides were deparaffinized in xylenes for two changes of 5 min and air-dried.

LCM

0
Fig. 1

The Auto PIX and Pix-Cell II E LCM system was used to microdissect cells from deparaffinized and dried tissue sections prepared as above. The laser beam was adjusted to melt the thermoplastic film in a spot of a diameter that visually corresponded to the diameter of the target cell. Captures were performed using 7.5- or 15-or 30 µm beam diameters according to cell size. Power settings were 50 or 40 or upto 100 mW, and laser pulse durations were 650 µs and 2.5 ms, respectively depending on the spot size. The success of harvest was evaluated by comparison of the image of cells captured on the cap versus the image of the tissue after removal of harvested cells. In general, the targeted plant cells were precisely removed from the section to the cap film, with nearly 100% efficiency, and without visible contamination with other cells. If additional material adhered to the harvested cells, they could be removed by blotting the post-harvest film with a Post-It adhesive strip (3M, St. Paul). Only cells immediately within the laser halo adhere to the thermoplastic film, and other cells are removed onto the Post-It strips. This treatment did not appear to influence the yield or quality of subsequent RNA extractions.

Figure 1 illustrates the process of laser-capture microdissection. A prepared tissue section is placed on the slide (1). After cells of interest have been located (2), a clear microfuge cap is suspended over them on the slide (3). An near-infrared laser beam is pulsed through the cap (4). The laser melts an ethylene vinyl acetate (EVA) layer on the cap. The EVA will dimple and adhere to the cellular target and the cap can be lifted (5). The isolated cells can now be processed (6).

LCM offers several advantages over other cell capture approaches:

  1. The near-infrared laser does not damage the target cells or any adjacent cells

  2. Multiple cell harvests can be performed from the same sample.

  3. The technique works with paraffin sections, so that blocks can be archived and resampled.

  4. Single cells, groups of cells and intricately shaped areas can be easily captured. In addition, the captured region can be inspected visually.

  5. A third-generation LCM instrument can also perform UV cutting.

RNA Extraction

Followed manufacturers protocol Picopure RNA isolation Kit (Arcturus).

RNA Quantification

RNA was measured fluorometrically on a microtiter plate reader (Wallac 1420, PerkinElmer Wallac, Turku, Finland). Samples were measured after 2 min and 5 min of incubation with the Ribogreen reagent (Molecular Probes, Eugene, OR) in black microtiter plates, with 485 nm excitation and 535 nm emission wavelengths, according to the manufacturer.

RT-PCR

RNA was reverse-transcribed using Sensiscript RT (Qiagen USA), primed with 1 µM oligo(dT) (18-mer, Ambion, Austin, TX) or 0.25 µM specific primer. PCR was performed with the Readymix RedTaq PCR Reaction Mix (Sigma-Aldrich), with 1 pmol µL1 of specific primers. The PCR conditions were adjusted based on the primers used.

Fig. 2

RNA Amplification

A T7 polymerase-based linear amplification systems, RiboAmp (Arcturus Engineering), was used according to the manufacturer. This proprietary system relies on forming double-stranded cDNA, followed by in vitro transcription by T7 polymerase. The manufacturer's quality control tests include the testing of amplified and unamplified probes on microarrays. The amplified probe signal matches the original RNA source, with a correlation coefficient of 0.91; duplicate amplifications have a reproducibility of r = 0.99 ( http://www.arcturus.com ).

Probe amplification and labeling

Harvested cells provide only very small amounts of total RNA, usually measured in nanograms. This makes it necessary to perform two rounds of linear amplification into mRNA-based amplified RNA (aRNA).

There are several important issues associated with aRNA probes:

  1. Amplified RNA (aRNA) is antisense to mRNA, so probes are directly labeled rather than being reverse transcribed into labeled cDNA.

  2. RNA-DNA hybridization tends to be less efficient than DNA-DNA. For this reason, we reoptimized hybridization conditions.

  3. RNA probes are subject to degradation, so it becomes necessary to eliminate or inhibit RNases.

Rice whole genome oligo arrays

0
Fig. 3

The NSF Atlas will rely upon community oligo arrays that are under development through another NSF project (Pamela Ronald, UC Davis). These arrays are based on data from community sequencing projects.

For the period prior to when these arrays become available, we have provisional permission to use an existing 2-slide whole genome 70mer array containing the following oligo set:

Total in set 58,404
Map to 10/03 genome 55,416
Unique genes 55,791
Match 10/03 predicted genes 21,186
Match only old predicted genes 4,125
Match to predicted japonica genes 94%
Fig. 4

  1. 30,480 expression-confirmed unigenes consisting of:

    1. the 16,611 japonica full-length cDNA oligos described in the Kikuchi Science paper.

    2. 13,869 additional EST clusters (mostly from japonica)

  2. 25,311 predicted unigenes from the corrected and update indica sequence (PLOS Biol., in press)

Figure 4 presents summary details for the oligo array:

We can see a sample hybridization slide in Figure 3. This is slide A of the two-slide set; it was hybridized with probes derived from total RNA samples of rice panicles (red) and cultured rice cells (green).

For oligo hybridizations, the following conditions are necessary:

  1. We need improved pre-hybridization in order to block non- specific probes.

  2. We need to provide more probe purification than is usually the case.

  3. A hot start is need to enhance the specificity of the hybridization.

Data collection

Scanning:

  1. Two-slide sets are scanned on a GenePix 4200A scanner.

  2. Initial analysis of the slides is performed using the GenePix 5.0 software packaged.

Replicates:

  1. Three biological repeats are collected for each cell type.

  2. Independent hybridizations are performed using a dye swap.

  3. The correlation coefficient between two repeats is calculated and values lower than 0.85 are rejected.

  4. Normalization is performed among different cell types. This ensures that profiles are comparable among all cell types.

  5. Cell types are analyzed on a 'first in first out' (FIFO) basis. Cell types from the same organ are grouped for analysis.

Normalization:

  1. The locally weighted scatter plot among repeats is smoothed using Lowess. This is to reduce the impact of noise caused by dye swapping.

  2. We then perform quantile normalization so that the same data distribution exists among repeats of each cell type.

  3. Finally, we perform median-based normalization among different cell types to normalize expression intensities.

Gene expression:

  1. We rely on an intensity-based determination of transcription.

  2. Recognition of spots is performed using GenePix tools. Bad spots are manually flagged.

  3. An expression cut-off is drawn for each hybridization based upon these negative control spots.

  4. To be considered as expressed, a gene should be detected at least twice in three separated hybridizations.