The BioLiqX cf-RNA Isolation QC Kit contains all reagents and primers required for the detection of three “house-keeping” miRNAs including hsa-miR-16, hsa-miR-21 and hsa-miR-451a, as well as a spike-in control cel-miR-39 in any RNA sample using RT-qPCR. The key features of the method are highly efficient polynucleotide tailing and simultaneous reverse transcription of all short RNA species in a sample which are followed by real-time PCR amplification. The tailing and reverse transcription (TaRT) mix included in the kit is an optimized composition of enzymes, nucleotides, mineral salts, and RT primers that secures an efficient conversion of highly diluted miRNA molecules into cDNA in a single reaction. In the second step, the cDNAs serve as templates for ultra-sensitive and low-background real-time PCR reactions with Green DNA Dye (a SYBR® Green analog) and miRNA-specific primers. Due to its simplicity, the procedure secures low variation between technical replicates, short hands-on time, and easiness of automation. High specificity and sensitivity of qPCR are achieved by optimal concentration of nucleotides, mineral salts, Green DNA Dye and Hot Start Taq DNA Polymerase in the reaction. All reactions can be set up at the room temperature without the risk of non-specific amplification. The BioLiqX cf-RNA Isolation QC kit is ideal for monitoring the RNA isolation efficiency between samples and has a similar sensitivity as compared to analogous preamplification-free miRNA assays.
The kit is delivered as a standard cardboard microtube box containing ready-to-use TaRT Mix for tailing and reverse transcription, qPCR Mix for Real-Time PCR reaction, as well as four vials with miRNA-specific primers: hsa-miR-16 PM, hsa-miR-21 PM, hsa-miR-451a and cel-miR-39 PM. The kit also includes synthetic cel-miR-39 spike-in control. The supplied volume of the BioLiqX cf-RNA Isolation QC Kit is completely custom, however, the minimal size of the kit is for 24 samples.
On the first step, a polyA-tails are added to 3’-OH termini of all RNAs and cDNA is synthesized simultaneously using an anchored dT-rich primer with custom 5’-terminal sequence. Finally, cDNA is amplified using miRNA-specific forward and reverse primers during qPCR reaction, while Green DNA Dye (a SYBR® Green analog) is used for the detection and quantification of dsDNA amplicons in real-time (see figure below).
Please note that preamplification-free protocols for miRNA detection with RT-qPCR have a fundamental sensitivity limit due to a dilution factor. Thus, an RNA sample becomes typically diluted 2-3 fold in a cDNA reaction initially and another 6-30 fold before the qPCR (depending on the kit supplier). Finally, the cDNA product suffers additional 5-20 fold dilution in a subsequent qPCR reaction. To negate the dilution effect, highly-sensitive preamplification-based BioLiqX HS miRNA Assays are available as a separate kit.