High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The overall goal of this procedure is to profile micro RNA expression efficiently, inexpensively, and with minimal starting material by combining multiplex quantitative R-T-P-C-R with a microfluidic platform. This begins with concentrating extracted RNA, followed by multiplex reverse transcription and multiplex pre amplification. The next step is to purify away excessive primers.

Finally, singleplex micro RNA expression is profiled using a microfluidic platform. The result is accurate and highly reproducible micro RNA expression levels across samples of interest. The main advantage of this technique of existing methods is that the additional purification step after pre-EMP amplification improves the accuracy and reproducibility of the multiplex Q-R-T-P-C-R method.

Begin with RNA that has been extracted from serum. Using an RNA purification kit or phenol chloroform based RNA method from homogenized tissue like triol, all extractions must be followed with a wash step and all storage is done at minus 80 degrees Celsius. Concentrate the RNA solution.

If starting with a limited amount of sample, such as from serum first, insert a MicroCon sample reservoir into a vial, then pipette the RNA solution into the reservoir, being very careful to not touch the membrane with the pipette tip. Now position the vial and filter in the centrifuge with the crack band towards the rotor and run the centrifugation for about three hours. After three hours, the RNA will be fully concentrated on the membrane to elute the RNA.

Add a desired amount of RNAs free water to the membrane, invert the reservoir and place into a new vial. Then spin for three minutes at four degrees Celsius with maximal 3000 times G.Store the concentrated RNA at minus 80 degrees Celsius until needed After performing an RT reaction on the RNA using xpl reverse stem loop primers, followed by 12 to 18 rounds of PCR cycles. The microRNAs are represented by CD NA strands.

This is called the pre PCR product. Referring to the upcoming QPCR. The pre PCR product needs to be purified on a 10%poly acrylamide gel alongside a 10 base paired DNA ladder load each lane with 5.5 microliters of six x orange G combined with 27.5 microliters of pre PCR product.

Run the gel at 150 volts for 55 to 60 minutes. Romo phenol blue runs on this gel in the 20 base pair region. After the gel has run, stain it with cyber gold for 25 minutes, unexposed to the light and on a shaker from the stained gel.

Cut out the pre PCR product band from 60 to 80 base pairs and transfer the gel block to labeled tubes. If the product concentration is low, it may not be visible, but will still be in the gel. Now crush the gel band using tube in tube centrifugation and add 300 microliters of TEN buffer.

Incubate the buffer gel mix for four hours at 37 degrees Celsius with agitation. After four hours, transfer the fluid to a new tube and add 300 more microliters of TEN buffer. Incubate this mix overnight at four degrees Celsius with agitation.

The next day, transfer the tube contents again and proceed with an ethanol precipitation of the pre PCR product. When a chip is opened from its package, it must be primed within 24 hours and then loaded within 60 minutes of priming. Begin priming the array by injecting 150 microliters of control line fluid into each of the accumulators on the chip.

Be careful not to spill any fluid on the chip or inlets, or the chip will become unusable. Then place the chip in the integrated fluidic circuit or IFC controller and run the controller's priming script. As soon as the program begins, start preparing the samples.

Begin by preparing the pres sample Mix a combination of tack man, universal master mix, and loading reagents. Then using the 96 well loading format, combine 2.25 microliters of each sample with 2.75 microliters of pres sample mix to collect the loaded fluid. Briefly vortex and quickly centrifuge the plate at four degrees Celsius.

The sample plate is now prepared. Now use a new 96 well plate to prepare 13 x concentrations of the assay mixture. This is done by mixing one universal primer, forward primer and gene specific tac man probe per well.

The one X concentrations are one micromolar for the universal and forward primer and 0.2 micromolar for the tac man probe. Then add enough tween for a final concentration of 0.25%tween in a final well volume of five microliters. Mix well avoiding bubbles and briefly spin the plate to collect the fluid.

This is the assay plate. Begin the 96.96 Q-R-T-P-C-R by loading the chip. First load the samples and then the assays with the final volume of five microliters per inlet to the IFC.

Second, run the load mix script to load the samples and assays into the chip. When the script is over, peel back the protective film from the loaded chip and remove any dust particles or debris from the chip surface using adhesive tape. Now set up the bio system amplification program.

Load the chip into the system and run the program. The results can be analyzed with fluid dime software to provide amplification curves, whole chip heat maps and CT values for each. Well using pre PCR products, purified by size separation on native poly acrylamide gels, wild type DG CR eight and ER null mutant.

RNA was prepared compared to previous protocols. More microRNAs and a loss of false positive signals occur in both DGCR eight null and ER null backgrounds thanks to the purification step. In addition, the modified QR TPCR R approach allows for the proper categorization of rare D gcr eight independent dicer dependent small RNAs.

Here, 48 cera from patients and controls were screened for expression change of 384 different microRNAs. The data were normalized for each sample by subtracting the corresponding median and without using a spike in the controls. After watching this video, you should have a good understanding of how multiplex Q-R-T-P-C-R on a flu line platform can be used to simultaneously profile hundreds of micronase across many samples, even when starting with very limited input, RNA.