Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

The overall goal of this procedure is to measure the absolute levels of Plasma MicroRNAs using Quantitative Real-time Reverse Transcription PCR. This method can help assess the quantity of plasma microRNAs, even if their expression level is low. The main advantage of this technique is that the measured data can be compared with others from different studies or laboratories.

The implications of this technique extend to the samples from other species because a standard curve can be generated by using the synthetic RNA or the nucleotide. This method can provide insight into translational research to investigate promising safety biomarkers. First, thaw the frozen plasma samples obtained from the femoral vein of the Cynomolgus Monkeys on ice.

Transfer the Lysis reagent in Chloroform on ice for chilling before RNA extraction. Next, add 1, 000 microliters of Lysis reagent to 200 microliters of the sample. And vortex for one minute to ensure proper mixing.

Then add five microliters of five nanomolar synthetic Caenorhabditis elegans microRNA and 200 microliters of chloroform to the sample. Vortex the sample for one minute to mix properly. Then, place the sample on ice for two to three minutes.

Next, centrifuge the sample at 12, 000 G, for 15 minutes at four degrees Celsius. Next, carefully transfer 650 microliters of the aqueous phase to a new microtube. Then, add 975 microliters of Ethanol to the aqueous phase and pipette several times to ensure complete mixing.

Transfer the sample into the corresponding column and adapter and then vacuum dry for three minutes using vacuum manifolds. Then add 200 microliters of Ethanol to the column and vacuum dry for one minute. After vacuum drying, add 800 microliters of RWT Buffer to the column and, again, vacuum dry for two minutes.

Then add 800 microliters of RPE Buffer to the column and vacuum dry for two minutes. Add the RPE Buffer twice, followed by vacuum drying. Next, add 300 microliters of Ethanol to the column.

After adding Ethanol, vacuum dry for one minute. Then, transfer the column in a new microtube and centrifuge the same at 12, 000 G at room temperature for one minute. After centrifugation, transfer the column to a new microtube and add 50 microliters of Nuclease-free water to it.

Let the column stand at room temperature for three minutes and then centrifuge at 8, 000 G at room temperature for one minute. Finally, store the eluate at minus 80 degrees Celsius until use. To prepare a synthetic RNA Oligonucleotide Concentration that corresponds to the target microRNA, dilute the stock solution 10-fold to achieve a working solution with the highest concentration to plot the standard curves.

Next, to constitute a multiplex pool of reverse transcription primers, mix equal volumes of the 20-strength primers for target microRNAs. Then, prepare the reverse transcription reaction mix. Then add 10 microliters of reverse transcription reaction mix to five microliters of RNA sample.

Mix the two by pipetting several times. Then, incubate the mixture on ice for five minutes. After placing the sample in the Thermocycler, start the cycle.

Once the program is over, store the reverse transcribed sample at minus 80 degrees Celsius. To constitute the multiplex assay primer pool, add equal volumes of five microliters of assay primers corresponding to target microRNAs into the tube containing Tris-EDTA buffer in the final volume of 1, 000 microliters. Next, constitute the pre-amplification reaction mix.

Once the reaction mix is done, transfer 22.5 microliters of pre-amplification reaction mix to two point five microliters of reversed-transcribed sample. Then, pipette to mix the sample and pre-amplification master mix thoroughly and incubate on ice for five minutes and then leave the PCR tubes in the Thermocycler and start the run. Once the program is over, transfer all the samples at minus 80 degrees Celsius.

After thawing the pre-amplified or non-pre-amplified samples, dilute it five-folds with sterile water. To generate the standard curve, do 10-fold serial dilution of the samples derived from synthetic RNA oligonucleotides. Then, constitute the quantitative PCR reaction mix in a tube kept on ice.

Then add 18 microliters from the quantitative PCR reaction mix to the Fast Optical 96-Well Reaction Plates. Then add two microliters of the diluted samples into the wells. After sealing the plate with adhesive film, briefly centrifuge the sample at 500 G for 15 seconds.

Then start the real-time Thermocycler program. Use a SDS software Version 2.4 that functions with the corresponding real-time thermal cycler to compute the raw copy number of each sample. Then calculate the absolute copy number from the raw copy number using Excel software.

Amplification efficiency of miR-122, miR-192, and miR-133a was analyzed by plotting standard curves, which explains the relation between the log concentration versus the quantification cycle for the non-pre-amplified samples. The standard curves for miR-122, miR-192, and miR-133a show a linear relationship between quantification cycles and log concentration of the samples. Next, amplification efficiency of miR-1, miR-499a, and miR-206 was analyzed by plotting standard curves for pre-amplified samples.

To analyze the amplification efficiency, the slope was computed using linear regression. The slope in standard curve A shows the absence of specific amplicons at 1, 000 copies per microliter concentration for miR-499a and miR-206. However, the non-specific amplicons were obtained at 1, 000 copies per microliter for miR-1.

Here, non-pre-amplified and pre-amplified miR-206 samples were used in duplicates to derive the amplification plots. The quantification cycle values for non-pre-amplified and pre-amplified miR-206 was estimated to be 39.9, plus or minus one point nine and 27.0, plus or minus two point two, respectively, representing almost similar values between the duplicates as shown in the plots. Next, plasma microRNAs obtained from Cynomolgus monkeys were subjected to profiling to compute their absolute values.

The dot plots, obtained from microRNA profiling representing their expression levels, shows that miR-122, miR-133a, and miR-192 is detectable without pre-amplification. Whereas, miR-1, miR-206, and miR-499a will require pre-amplification due to low expression levels. After watching this video, you should have a good understanding of how to measure the absolute levels of plasma microRNA, using RT-qPCR.

And pre-amplification;it's useful to improve detection with small quantities of microRNAs.