BioLiqX HS miRNA qRT-PCR Assay (Customized)

Description

Customized BioLiqX HS miRNA qRT-PCR Kit for High Sensitivity miRNA Detection

• Pre-amplification step and negation of dilution effect ensure superior sensitivity
• Perfect for low copy number circulating miRNAs in biological fluids, such as plasma, and in exosomes/extracellular vesicles
• High specificity for any miRNA(s) of your choice
• Ideal for validating Small RNA-Seq results quantitatively
• Based on CATL technology: BioLiqX Small RNA-Seq results can be validated directly on the same libraries using BioLiqX HS miRNA qRT-PCR Assays

Workflow of the BioLiqX HS miRNA qRT-PCR Assay

Customized BioLiqX HS miRNA qRT-PCR KIts can be produced for you for any miRNA including truncated miRNAs and isomiRs as well as any other short RNA fragment.

The kit utilizes a single-tube protocol for the 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. Subsequently, 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. 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 biological fluids but can be also applied to any other 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 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. 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.

Kit Contents and Customization:

The kit 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 vial(s) with miRNA-specific primers for any miRNA(s) of your choice. By default, the kit also includes synthetic cel-miR-39 spike-in control.

The supplied kit size of Custom BioLiqX HS miRNA qRT-PCR 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.

please contact us for a quote and let us know the miRNAs you like to detect and the desired kit size!