Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

The overall goal of the following experiment is to demonstrate three different probe based real-time PCR methods used for quantifying micro RNA in plasma and serum samples. Probe based real-time PCR is achieved by hydrolysis and detection of the fluorescently tagged probe, which contains a fluorescent reporter and a quencher. When the tack polymerase extends from the upstream primer and reaches to the five prime end of the probe, it leads to a physical dissociation of the fluorescent emitter from the quencher.

Next, a single molecule of fluoro four is released, which is recorded by the detector and presented as an incremental increase in the fluorescent signal from that reaction. Ultimately, the amount of PCR product generated can be measured based on the increase in fluorescence, allowing an accurate quantification of the amplified target amplifi. This method is emerging as a promising method for discovering novel biomarkers for various cancers and other diseases such as diabetes.

Though this method can provide insight into the abundance of microRNAs in serum and plasma, it can also be applied to other samples such as peripheral cells and tissues. A visual demonstration of this protocol is critical as some of the steps involved in sealing the nano fluidics array slide can be difficult to learn by simply reading the protocol For probe based real time quantitative PCR mature micro RNA detection at 4.2 microliters of the individual QPCR reagent mixes to each well of an optical 96 well plate, followed by 0.8 microliters of the appropriate freshly prepared CDNA reaction solution. Seal the plate with the appropriate optical cover and then start the QPCR on the real-time PCR system.

To prepare a microfluidics array card with one to 1000 nanograms of total RNA starting input first thaw the pool A and B primers buffer, deoxy nucleotide phosphates, magnesium chloride, and RNAs inhibitor on ice. Then to perform reverse transcription, aliquot 100 nanograms of each RNA sample into a pool A or pool B tube, followed by the addition of the appropriate volume of nuclease free water to bring the total volume in each tube to three microliters. Next aliquot 4.5 microliters of the appropriate reverse transcription reagent mix into the appropriate corresponding tubes.

Then place the samples into the thermocycler and start reverse transcription. While reverse transcription is progressing. For total RNA sample inputs between one and 350 nanograms thaw the predefined pool of pre amplification primers on ice when the samples are ready, aliquot 22.5 microliters of the appropriate preamp reagent.

Mix into the pool A and pool B tubes and add 2.5 microliters of the reverse transcribed CDNA sample into each. Then load the samples into the thermocycler and start the preamp cycle. At the end of the cycle, add 75 microliters of 0.1 x tris EDTA buffer to the pre amplified CDNA to load the microfluidics card.

Next, add 450 microliters of QPCR reagent mix to nine microliters of the diluted PREPL amplified CDNA for card A, use the PREPL amplified product prepared using the primer pool A and add 441 microliters of nuclease free water to a final volume of 900 microliters. Then once it reaches room temperature, remove the tack man low density array card from its packaging and place it foil side down on a clean area. Add 100 microliters of PCR reaction.

Mix into each of the eight fill ports on the card. Spin the card for one minute at 331 GS and room temperature. Then in a slow, steady and single uniform stroke, push the carriage across the card until it reaches the endpoint of the sealer.

Remove the sealed array card and cut off the reservoirs. Then confirm that the correct block heated lid and sample carrier are installed in the microfluidics array realtime PCR system. To load the nano fluidic arrays, thaw the diluted prepl, amplified CDNA and tack man realtime QPCR reagent.

Mix and mix the QPCR reagent bottle by swirling. Next, add 22.5 microliters of QPCR reagent mix to 22.5 microliters of diluted prepl, amplified CDNA. Then add five microliters of each sample into eight wells of the nano fluidics array workflow 384 well sample plate taking care to place the pool A and pool B of each sample.

In adjacent eight well blocks when all the samples have been transferred, cover each section of the plate with pieces of open array sample plate sealer. Then thaw the first nano fluidics array slide to room temperature after approximately 15 minutes. Confirmed that the correct block edit lid and sample carrier are installed in the nano fluidics array system, and then loosen the plunger of the immersion fluid syringe with gentle pulling.

Remove the cap, put the tip in place and flush the air from the tip. Then place the loading system tips within the loading system. Open the lid and place the sample plate into the loading system.

Now put on a pair of tightly fitting gloves. Carefully open the slide packaging and slowly tip the slide out of the package without touching the top. Place the slide into the loading system with the barcode on the left.

Then remove the sealer from the portion of the sample plate intended for loading, and use the loading system software to enter the slide barcode slide position, sample position and tip configuration. While the slide is loading, remove the clear and red plastic from the bottom of the slide lid. Then carefully remove and seal the slide within 90 seconds of the end of the loading process.

Place the slide within the plate clamp and the slide lid onto the slide for 30 seconds confirming that the lid is positioned so that barcode is correctly displayed. Then position the immersion fluid syringe within the slide so that the tip is pressing against the lid and slowly fill the slide with immersion fluid taking care that the fluid runs along the lid. Once the slide is full, seal it with the plug, turning the screw until the handle breaks off.

Finally, remove the plastic cover on the top of the slide lid and carefully place the slide into the slide carrier of the real-time PCR system. Take care to support the bottom of the slide as it is being lowered without touching the top of the slide. Then initialize the PCR system and start the quantitative PCR program within one hour of loading the samples.

Using this procedure, a reaction volume of five microliters can produce results similar to those achieved using a 20 microliter volume with a strong correlation. Up to 39 cycles here, bland Altman and correlation plots are shown for all 754 micro RN tested on the same sample using microfluidics array cards as just demonstrated microRNAs that have CT values between zero and 19.99 in both trails also exhibit similar expressions with a coefficient of determination of 98%Further, the number of microRNAs with significant differences observed between the trials increases when CT values between 30 and 40 are selected. In this graph, the nano fluidics array platform data for all 754 microRNAs is shown.

It is important to examine the amplification curves to ensure that the results are indicative of true amplification. Housekeeping microRNAs, such as U six, should also be examined for variability. For example, in this graph, a typical clustering of U six replicates displaying low standard deviation are shown indicating the reliability of the data.

Finally, these data illustrate the increased variability of U six replicates in a second sample. High throughput probe based technologies such as the microfluidics and Nana fluidics arrays offer the ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a very short time. Each of the workflows are designed to cater to different throughputs based on the number of microRNA targets and the number of samples to be analyzed.

After watching this video, you should have a good understanding of how to perform different probe based real time PCR techniques for majoring micro NA abundance.