BioLiqX HS miRNA Assays

Overview

BioLiqX HS miRNA Assays include all reagents and primers required for the simultaneous qPCR detection of “house-keeping” miRNAs hsa-miR-16, hsa-miR-21 and hsa-miR-451a, a spike-in synthetic control cel-miR-39, as well as miRNAs which proved themselves as promising extracellular circulating biomarkers in multiple peer-review research publications. Currently, the latter list includes: hsa-miR-122 (Liver-specific), hsa-miR-208a (Myocard-specific), hsa-miR-208b (Myocard-specific), hsa-miR-499 (Myocard-specific), hsa-miR-133a, hsa-miR-206, hsa-miR-9, hsa-miR-124, hsa-miR-150, hsa-miR-216, hsa-miR-375, hsa-miR-192, hsa-miR-30, hsa-miR-200a, hsa-miR-200b, hsa-miR-200c, hsa-miR-141, and hsa-miR-429. Besides, we offer assays for custom miRNAs (including truncated miRNAs and isomiRs) as well as any other short RNA fragment.

The kit utilizes a single-tube protocol for the prior conversion of all short RNA molecules into a preamplified cDNA library by adding certain reagents and enzymes to the RNA sample in a sequential manner. Afterward, the preamplified cDNA serves as a template for ultra-sensitive and low-background real-time PCR reactions with Green DNA Dye (a SYBR® Green analog) and miRNA-specific primers. Apart from the preamplification reaction, high specificity and sensitivity of BioLiqX HS miRNA Assays are achieved by the optimal concentration of nucleotides, mineral salts, Green DNA Dye and Hot Start Taq DNA Polymerase in the final qPCR reaction. All reactions can be set up at room temperature without the risk of non-specific amplification. The BioLiqX HS miRNA Assays are ideal for detecting low copy number circulating miRNAs in the biological fluids but can be also applied to any sample. The whole procedure can be completed within approximately 8 hours and requires typically a hands-on time between 30-60 minutes depending on the number of samples.

The kit is delivered in a standard cardboard microtube box that includes: (1) reagents for cDNA synthesis and preamplification (Tailing Buffer, Tailing Nucleotides, Tailing Enzyme, Ligation Buffer, Ligation Enzyme, RT Mix, RT primers and PreAmp Mix; (2) qPCR Mix for Real-Time PCR reaction and vials with miRNA-specific primers (hsa-miR-XXX PM). By default, the kit also includes synthetic cel-miR-39 spike-in control. The supplied volume and the exact composition of BioLiqX HS miRNA Assays can be completely customized depending on your research goals. However, the minimal volumes of the reagents used for the initial preamplification (library generation) are for 24 samples. Besides, the miRNA primers are compatible with the NGS libraries generated by BioLiqX Small RNA-seq Kit. Therefore, small RNA sequencing results can be validated directly on the same libraries using qPCR Mix and miRNA primer pairs from BioLiqX HS miRNA Assays.

Method Workflow

The BioLiqX HS miRNA Assays are based on the so-called “Capture and Amplification by Tailing and Ligation (CATL)” approach for cDNA synthesis and pre-amplification of short or fragmented RNA in the original sample before the final Real-Time PCR step. Briefly, miRNA is subjected to a polyadenylation reaction followed by the ligation of 5’-adapter. The input RNA flanked by 5’-adapter and 3’-poly(A) tails is then converted into cDNA using anchored RT primer carrying poly(T)-rich sequence and custom 3’-adapter sequence. The cDNA is then PCR amplified (typically for 10-14 cycles) using primers carrying terminal sequences matching the 5'- and 3'-adapters. Finally, the pre-amplified cDNA is used as input for Real-Time PCR reaction with miRNA-specific forward and reverse primers. The Green DNA Dye (a SYBR® Green analog) is used for DNA quantification during Real-Time PCR (see figure below).

The BioLiqX HS miRNA Assays allow detection of very short cDNA fragments including truncated microRNAs and isomiRs. In addition, pre-amplified libraries generated by the protocol can be used for the simultaneous qPCR analysis of various other cell-free RNA fragments using custom primer pairs.

Example of Assays Performance

BioLiqX HS miRNA Assays have superior sensitivity as compared to preamplification-free protocols mainly due to negating RNA and cDNA dilution effect during RT-qPCR. The figure below shows the benchmarking of BioLiqX HS miRNA hsa-miR-16, hsa-miR-21 and cel-miR-39 assays with the corresponding preamplification-free assays from either BioLiqX cf-RNA Isolation QC kit or two competing research kits.

Figure 1. Benchmarking of BioLiqX HS miRNA assays with preamplification-free protocols. Specifically, total RNA was isolated from 0.4 mL of human blood plasma with 0.5 pg 22 nt cel-miR-39 control premixed after the initial lysis. The RNA was eluted in the total volume of 50 µL, and 5 µL of eluates were taken as inputs for each reaction (in triplicates). The BioLiqX HS miRNA libraries were subject to 12x pre-amplification cycles and diluted 10-fold in nuclease free water. The cDNA generated by BioLiqX cf-RNA Isolation QC kit and the competing kits was diluted 6-fold. The proportions of the diluted cDNA in the final qPCR reactions were equal for each protocol. The Real-Time PCR was performed using LightCycler 480 Real-Time PCR System (Roche) according to the recommendations of each kits' manuals. Raw threshold cycle (Ct) values were determined by the second derivative max method. Each bar represents the mean (SD) of three replicates.