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miRNA probes for cancer diagnostics – What BioGenex offers?

miRNA biology

microRNAs or miRNAs are small, ~21-24 nucleotides long, specialized, ubiquitously expressed and evolutionary conserved cellular RNAs that have numerous roles in cellular functions ranging from developmental and stem cell to aging, immunity and cancer. First discovered in nematodes, it has been found in almost all classes of organisms wherein they function as part of cellular networks cross-talking with multiple target mRNAs, thus affecting post-transcriptional machinery of the cell. MicroRNAs are precisely regulated (both spatially and temporally) in these cellular functions. Recently, microRNAs have been touted as promising agents in clinical diagnostics and therapeutics especially in the cancer domain.

miRNA and cancer

Deregulated expression of miRNAs has been shown to be prevalent in most of the cancers, sometimes as oncogenic drivers and at other times acting as tumor suppressors. Hence, expression profile of microRNAs has been considered as a better indicator of genomic aberration than their target mRNA. These microRNAs exhibit tissue or cell lineage specificity right from developmental or embryonic stage that can be utilized as a cell or tissue-specific marker. Their smaller size and resistant to RNAse degradation render them superior to mRNA while assaying for expression profiling for cancer diagnostics [1, 2].


miRNA in cancer diagnostics

miRNAs can be utilized as potential diagnostic biomarkers in several ways:

  • Analysis of single nucleotide polymorphisms (SNPs) in miRNA genes or miRNA binding sites in target mRNA or in miRNA processing machinery genes to predict cancer predisposition [1]. Additionally, these SNPs can be used as prognostic/predictive markers in patient management to assess the risk of treatment outcome.
  • Use of miRNA-based classifiers to identify tissue of origin for cancers of unknown primaries (CUPs). CUPs are currently one of the most challenging cancers to treat effectively as the primary site of origin of cancer remains undiagnosed.
  • Utilizing a standard set of altered miRNA expression profiles to differentiate normal and cancer tissue by a comparative approach. Sub-typing different categories of cancer using standard panel of miRNAs that differentiates between the cellular types. The developmental profile of the tumor type is reflected in miRNA expression profile: a blind study of 22 different tumor types showed miRNA expression classified tumors according to tissue of origin with accuracy higher than 90%. Distinct types of cancers like leukemia, prostate cancer, epithelial cancer, basal-like cell cancer, HER2-breast cancer have been classified and sub-classified using miRNA markers in developmental cell lineage markers. Even in prognostic assays, miRNA-based tests are an alternative to mRNA/protein expression. miRNAs have been shown to have a role in endothelial recruitment in metastatic outcome of specific cancers like breast and endometrial cancer to various treatment regimens.
  • Early detection of cancers bypassing the invasive procedures by profiling circulating blood (serum) or tumor-derived biomarkers.

Please refer to table in [2] for an extensive analysis of miRNA based approach to precision medicine.

miRNA in tissues - in situ hybridization (ISH)

in situ analysis of miRNA in cancer tissues can be used as a diagnostic/prognostic assay. Due to its cellular localization and spatial information, in situ can give a better contextual analysis than other RNA assays like qRT-PCR or microarray-based methods which tell us simply the presence or abundance of a specific miRNA in a cell lysate. Specific examples where ISH-based miRNA analysis has been carried out are: miRNA-21 in colon adenocarcinoma, gliomas, pancreatic and breast cancer, miR-10b in pancreatic ductal adenocarcinoma and miR-221 in breast cancer [3]. Additionally, multiplex analysis of different miRNAs can be carried between different types of cancers. Designing accurate and stable miRNA probes that offer both specificity and sensitivity is of paramount importance in harnessing the advantages of ISH-based miRNA analysis in cancer diagnostics.

BioGenex miRNA probes

BioGenex has developed proprietary Super Sensitive Nucleic Acid (SSNA) miRNA probes that enhance signals from intrinsically low populated miRNAs. The SSNA oligos are synthesized using specially designed bases to give super high melting temperatures. The high melting temperatures enable stringent washes at elevated temperatures to remove non-specific binding. BioGenex  miRNA probes are densely labeled with five FAM molecules that amplify the signal, giving intense stains. The high-density fluorophore-labeled SSNA probes combined with the BioGenex Super Sensitive™ Detection System aid in detecting even less abundant miRNA in the samples.

Distinct advantages of probes:

  • These probes are super sensitive because of multiple labels – 5 FAM molecules are attached to these probes. FAM is universal and FAM antibody works well with BioGenex HRP-based detection system. Experiments have indicated that this is compatible with other dyes such as Hex, Cy3 and Cy5.5
  • Each probe has 2-4 special bases that make it heat-resistant even under high melting temperatures. This aids in more stringent washes under high temperatures to remove non-specific binding and thus enhance specificity of the probe.

Panel of probes as biomarkers

BioGenex miRNA portfolio includes over 235 unique miRNA probes, the ONLY commercially available extensive list of probes for miRNA ISH. 

These probes help to diagnose:

  1. Cancer of unknown primary
  2. Undifferentiated and poorly differentiated tumors
  3. Classification of cancer subtypes
  4. Grading and staging of cancer

Furthermore, SSNA probes have been clustered into cancer-specific biomarker panels after validation:

  1. Renal cell cancer subtyping
  2. Glioma subtyping
  3. Gastric subtyping
  4. Non-small cell lung cancer differentiation
  5. Cervical squamous cell carcinoma differentiation
  6. Cholangiocarcinoma and Pancreatic Ductal Adenocarcinoma differentiation
  7. Triple-negative breast cancer differentiation
  8. Prostate Cancer (PC) and Benign Prostatic Hyperplasia (BPH) differentiation
  9. Wild Type and Mutated BRCA –breast cancer differentiation
  1. Hayes J, Peruzzi PP, Lawler S. MicroRNAs in cancer: biomarkers, functions and therapy. Trends Mol Med. 2014 Aug; 20(8):460-9.
  2. Detassis S, Grasso M, Del Vescovo V, Denti MA. microRNAs Make the Call in Cancer Personalized Medicine. Front Cell Dev Biol. 2017 Sep 22;5:86.
  3. Urbanek MO, Nawrocka AU, Krzyzosiak WJ. Small RNA Detection by in Situ Hybridization Methods. Int J Mol Sci. 2015; 16(6):13259-86.

Topics: BioGenex, miRNA

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