BioLiqX HS miRNA qRT-PCR Assay (Premade for organ and metastasis specific miRNAs)

Description

BioLiqX HS miRNA qRT-PCR Kit for High Sensitivity Detection of organ and metastasis specific miRNAs

• Based on CATL technology: Pre-amplification step and negation of dilution effect ensure superior sensitivity
• Highly specific detection of low copy number circulating organ and metastasis specific miRNAs in human blood plasma, and in exosomes/extracellular vesicles

Elevated levels of organ specific miRNA in blood plasma can serve as organ damage indicators.
The premade BioLiqX HS miRNA qRT-PCR Assay includes all reagents and primers required for the simultaneous qRT-PCR detection of the following organ specific miRNAs in plasma:

Myocard: hsa-miR-208a/b, hsa-miR-499
Liver: hsa-miR-122
Brain: hsa-miR-9, hsa-miR-124
Kidney: hsa-miR-30
Intestines: hsa-miR-192
Pancreas: hsa-miR-216
Skeletal Muscle: hsa-miR-133a, hsa-miR-206
Lymphocytes: hsa-miR-150

In addition, the kit also includes all reagents and primers required for the simultaneous qRT-PCR detection of miRNAs in plasma, which are described to be involved in tumor progression and/or metastasis, including hsa-miR-375, hsa-miR-200a/b/c, hsa-miR-141, and hsa-miR-429

The kit also includes all reagents and primers required for the simultaneous qRT-PCR detection of “house-keeping” miRNAs hsa-miR-16, hsa-miR-21, hsa-miR-451a, and cel-miR-39 as well as a spike-in synthetic control cel-miR-39.

Workflow of the BioLiqX HS miRNA qRT-PCR Assay

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.

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 the BioLiqX HS miRNA Assay is 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 Assay is ideal for detecting low copy number circulating miRNAs in biological fluids such as human blood plasma 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.

Kit Contents:
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 22 vials with miRNA-specific primers for the 18 organ and metastasis specific miRNAs described above as well as for 4 “housekeeping” miRNAs as listed above. The kit also includes synthetic cel-miR-39 spike-in control.
By default, the kit is supplied for 24 reactions.

Kit Customization
Any kit sizes larger than 24 rxns can be manufactured for you –
please contact us for a quote!