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Updated: May 6, 2026
Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR
Published on: January 22, 2014
Real-time PCR assay based on the differential expression of microRNAs and protein-coding genes for molecular
Ratika Kunder1, Rakesh Jalali, Epari Sridhar
1Corresponding Author: Dr Neelam Vishwanath Shirsat, PhD, Advanced Centre for Treatment, Research & Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai 410210 India. nshirsat@actrec.gov.in.
Background:
Medulloblastoma has recently been found to consist of 4 molecularly and clinically distinct subgroups: WNT, Sonce hedgehog (SHH), Group 3, and Group 4. Deregulated microRNA expression is known to contribute to pathogenesis and has been shown to have diagnostic and prognostic potential in the classification of various cancers.
Methods:
Molecular subgrouping and microRNA expression analysis of 44 frozen and 59 formalin-fixed paraffin embedded medulloblastomas from an Indian cohort were carried out by real-time RT-PCR assay.
Results:
The differential expression of 9 microRNAs in the 4 molecular subgroups was validated in a set of 101 medulloblastomas. The tumors in the WNT subgroup showed significant (P < .0001) overexpression of miR-193a-3p, miR-224, miR-148a, miR-23b, and miR-365. Reliable classification of medulloblastomas into the 4 molecular subgroups was obtained using a set of 12 protein-coding genes and 9 microRNAs as markers in a real-time RT-PCR assay with an accuracy of 97% as judged by the Prediction Analysis of Microarrays. Age at diagnosis, histology, gender-related incidence, and the relative survival rates of the 4 molecular subgroups in the present Indian cohort were found to be similar to those reported for medulloblastomas from the American and European subcontinent. Non-WNT, non-SHH medulloblastomas underexpressing miR-592 or overexpressing miR-182 were found to have significantly inferior survival rates, indicating utility of these miRNAs as markers for risk stratification.
Conclusions:
The microRNA based real-time PCR assay is rapid, simple, inexpensive, and useful for molecular classification and risk stratification of medulloblastomas, in particular formalin-fixed paraffin embedded tissues, wherein the expression profile of protein-coding genes is often less reliable due to RNA fragmentation.
Insights
A new microRNA assay accurately classifies medulloblastoma subgroups, aiding in risk stratification. This simple, inexpensive method is effective even with fragmented RNA from formalin-fixed tissues.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Medulloblastoma comprises four distinct molecular subgroups: WNT, Sonic hedgehog (SHH), Group 3, and Group 4.
- MicroRNA deregulation is implicated in medulloblastoma pathogenesis and can serve as a diagnostic and prognostic marker.
Purpose of the Study:
- To investigate the utility of microRNA expression profiling for molecular classification and risk stratification of medulloblastoma.
- To validate a microRNA-based real-time PCR assay for medulloblastoma subgrouping.
Main Methods:
- Real-time RT-PCR assay was employed to analyze microRNA expression in 44 frozen and 59 formalin-fixed paraffin-embedded medulloblastomas from an Indian cohort.
- Differential expression of nine microRNAs was analyzed across the four molecular subgroups and validated in a larger cohort of 101 medulloblastomas.
Main Results:
- A panel of nine microRNAs and 12 protein-coding genes accurately classified medulloblastoma subgroups with 97% accuracy.
- Specific microRNAs (miR-193a-3p, miR-224, miR-148a, miR-23b, miR-365) were significantly overexpressed in the WNT subgroup.
- Underexpression of miR-592 or overexpression of miR-182 in non-WNT, non-SHH medulloblastomas correlated with significantly inferior survival rates, indicating their potential as risk stratification markers.
Conclusions:
- A microRNA-based real-time PCR assay provides a rapid, simple, and cost-effective method for medulloblastoma molecular classification and risk stratification.
- This assay is particularly valuable for formalin-fixed paraffin-embedded tissues, overcoming limitations of RNA fragmentation affecting protein-coding gene expression analysis.
