Solutions for cell-free RNA analysis

• ISOLATION
• SEQUENCING
• REAL-TIME QPCR

Expertise and Key Technology

The research kits offered by Heidelberg Biolabs are based on in-house protocols and know-how in cell-free nucleic acids analysis developed in German Cancer Research Center (Molecular Epidemiology Group) and University Hospital Heidelberg (Division of Molecular Biology of Breast Cancer). Those laboratories have previously pioneered in the characterization of extracellular circulating miRNA and have more than a decade of research experience in the field of cell-free nucleic acids. One of our core methods includes "Capture and Amplification by Tailing and Ligation (CATL)", a highly efficient approach for cloning and preamplification of short RNA and DNA molecules from sources containing low nucleic acid content such as biological fluids, isolates of extracellular vesicles, forensic and archeological samples. The "CATL" procedure can be also applied for generating libraries for high-throughput sequencing as well as for enhancing the sensitivity of Real-Time qPCR detection.

RNA CATL principle

Fragmented single-stranded RNA molecules undergo polynucleotide tailing (e.g. polyadenylation) at the 3'-termini with certain ribonucleotides or ribonucleotide analogs and ligation of custom adapter at the 5'-termini. In the follow-up step, the RNA is converted into cDNA by reverse transcription in the presence of the anchored RT primer containing a sequence complementary to the polynucleotide tail and a custom 5'-terminal sequence. The second cDNA strand is generated during the first cycle of the final pre-amplifying PCR reaction. The polynucleotide tailing is a significantly more efficient way to capture 3-OH termini of single-stranded nucleic acids as compared to ligation of adapters and has little or no biases. As a result, DNA libraries produced by CATL approach have higher complexity as compared to the libraries generated by purely ligation-based methods,  Besides, CATL allows qPCR detection of single miRNA copies and customized massive parallel sequencing from picogram inputs of RNA.

Circulating miRNA Biomarkers

MicroRNAs (miRNAs) are short (18–24 nt) non-coding RNA molecules that normally regulate gene expression by repressing the translation and enhancing the hydrolysis of target mRNAs. However, after the death of the parental cells miRNAs remain stable in the nuclease-rich extracellular space for prolonged periods predominantly due to their association with proteins of the Argonaute family. Consequently, circulating miRNA profiles have been shown to reflect the ongoing pathogenic processes in the body’s tissues and organs, and enable highly sensitive non-invasive early diagnosis of multiple pathologies including various liver toxicity, myocardial infarction, metabolic syndrome, diabetes and brain ischemia. The practical utility of circulating miRNAs as biomarkers for longitudinal monitoring of pathophysiological processes in certain organs has been confirmed by a vast number of research reports. The scientists working for Heidelberg Biolabs have previously explained the mechanism of extracellular miRNA stability and developed ultrasensitive methods for the detection and characterization of such nucleic acids.

"Mi-Test"

One of our major goals is to establish a framework for preventive diagnostics of cardiovascular disorders, fatty liver, brain ischemia, diabetes as well as certain cancers using extracellular circulating miRNA biomarkers. Conceivably, such the measurement of organ-specific miRNAs (along with other circulating RNA species) could be performed in a separate blood test ("Mi-Test") at point-of-care stations along with conventional blood tests. The current version of "Mi-Test" prototype consists of several miRNAs expressed exclusively in the myocardial cells and whose presence in the blood plasma, therefore, is indicative of the ongoing cell death in the myocardium. In the future, such a test is planned to include other tissue-specific extracellular circulating RNA fragments which can serve as early indicators of ongoing pathological processes.