Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

This method is successfully optimized to classify breast cancer patients into known and not-known therapeutic groups. The high sensitivity of this assay identify heterogeneous samples that can be further studied using laser microdissection. The main advantage of the RNA-based multiplex assay is the reproducibility of results using Hematoxylin and Eosin-stained formalin-fixed paraffin-embedded archival material allowing high resolution outputs and biomarker validation.

Prepare the tissue as described in the text protocol. To begin the dissection, set the slide limits and calibrate the stage movement of the microscope. Now scan the stained membrane slides using the 4X objective.

Select and encircle the target areas for microdissection on the membrane slide. Keep track of the total area. At least 42 square millimeters is needed for adequate downstream RNA analysis.

Then initiate the laser cutting at the defined parameters. Next, use the cap-lift mechanism to retrieve the dissected section and set the section onto the diffuser cap of a labeled tube attached to a mechanical appendage. If the dissected section is not retrieved onto the cap, repeat the dissection of the target area.

Accuracy of microdissection is a critical step and one should always ensure that the microdissected area present on the cap coincides with the marked, scanned image. Collect as many tissue sections as desired, each in a separate tube, then proceed with lysing the tissue samples. For the lysis, apply 2.4 microliters of homogenizing solution pre square millimeter of tissue area in each sample, then vortex the samples for 10 seconds at the maximum speed.

Next, spin down the samples for five seconds at 2, 500 g. Then transfer the samples to 1.5-milliliter tubes if required, and place in a shaking heating block at 65 degrees Celsius. Shake them at 600 rpm for 12 to 18 hours.

Then centrifuge the lysates at 21, 000 g for five minutes at room temperature. Carefully collect the clear supernatant into a new labeled tube. Avoid transferring any tissue fragments.

The supernatants can be stored at minus 80 degrees Celsius. After preparing all the reagents for the hybridization-based assay, load up to 90 wells of a magnetic separation 96-well plate with 25 microliter tissue homogenate. Load 25 microliters of control samples to three designated wells and load a 25-microliter homogenizing solution to three wells as the blanks.

Then seal the magnetic separation plate using a clear plastic pressure seal and place the plate in a 54-degrees Celsius incubator for 18 to 22 hours with shaking. The next day, mount and lock the plate on a handheld magnetic plate washer, then remove the pressure seal and wait one minute for the magnetic beads to settle. Now perform a wash.

Drain the plate over a sink and use a tissue paper to gently blot it dry, then load all the wells with 100 microliters of 1X wash buffer and wait 15 seconds. Repeat this process three times to complete the wash step. After removing the last wash, reload the wells with 50 microliters of the pre-amplifier reagent, then seal the plate with an aluminum plate sealer and shake the plate at 800 rpm for one minute at room temperature.

Then continue shaking the plate at 50 degrees Celsius for an hour. When this is complete, perform another wash step. Next, reload the wells with 50 microliters of the amplifier reagent, seal the plate with an aluminum plate seal, and repeat shaking incubation of one minute at room temperature followed by an hour at 50 degrees Celsius, followed by another wash step.

Now vortex the label probe for 10 seconds at maximum speed and then add 50 microliters of label probe to each well. Then reseal the plate and repeat the shaking incubation as before, followed by another wash step. Next load the wells with 50 microliters of freshly-mixed Streptavidin phycoerythrin, then perform a shaking incubation at room temperature at 600 rpm, followed by a wash step using Streptavidin wash buffer.

After completing the wash step, load each well with 100 microliters of Streptavidin wash buffer and seal the plate with an aluminum plate seal, then shake the plate at 800 rpm at room temperature for three minutes. During the last incubation step, start up the magnetic bead analyzer and ensure the instrument is calibrated and validation protocols have been performed. In the software, add a new analysis by selecting Create Batch using an existing protocol from the Batches tab.

Next, in the plate layout, designate the wells as unknown or blank and provide a label for each in the Sample panel. Now remove the aluminum seal from the reaction plate, eject the plate loader tray, and load the plate into the analyzer. Next, click on Retract, then Run Batch to initiate the analyzer.

After the reading, eject the plate and clean the magnetic bead analyzer as recommended by the manufacturer. For analysis, go to the Batch Results tab. Select the respective analysis file and use the option to export it as a csv file, then create averages and raw scores using a spreadsheet software.

Ensure controls show the expected signals. Normalize the sample raw data and analyze the data distribution using a data science platform or using defined algorithms to execute a principle component analysis. The performance of the multiplex bead reader and assay is critical and running serial dilutions of well-annotated control samples prior to valuable patient samples ensures correct results.

For attributes, select the subset of normalized gene data and a nominal variable such as a breast cancer subtype. Apply a trained and validated classification algorithm to characterize unknown samples. The described method has been applied for the simultaneous measurement of 40 transcripts in H&E-stained, microdissected, highly degraded FFPE material.

Using this method, it is possible to show accurate characterization of receptor status and differential expression of the mesenchymal marker FN1 when comparing tumor and matched control tissue in the various receptor positive and negative subtypes. Heterogeneity within tumors can be characterized using this method. An ER positive tumor exhibits spatial differential expression of FN1 within areas showing distinct tumor morphology and mitotic activity.

After watching this video, you should have a good understanding on how to measure multiple genes directly from tissue lysates following hybridization of target RNA to beads, signal amplification, and quantification. Assay sensitivity allows the use of laser microdissected material. While attempting this procedure, it is important to remember to calibrate the equipment prior each assay and to determine the sensitivity range using quality control material before running precious patient samples.

The assay workflow is easily adaptable to clinical laboratories. RNA profiling of up to 90 samples can be performed in 12 hours over two days. Measuring breast cancer receptor status in a pathology lab is time-consuming and requires experienced pathologists.

Using this digitalized, multiplex bead-based assay quantifies RNA expression accurately. Analysis of fractional biomarkers in biopsies in a primary diagnosis is a presentation of the tumor. Using this methodology with a wider biomarker panel and also utilizing whole sections identifies heterogenic samples.

Development of assays using this methodology is versatile and requires low input of sample. Sample include clinical annotated samples important for biomarker validation and also liquid biopsies which are important for patient surveillance and early diagnosis.