Introduction
microRNAs (miRNAs) have been shown to play an important role in regulation of gene expression in a variety of diverse biological processes throughout the animal and plant kingdoms. Increasingly, researchers are focusing on the causes and consequences of changes in miRNA expression in both normal human development and disease. In the field of molecular oncology, profiling of miRNA expression in cancer cells offers the possibility of new insights into the causes of cancer and the potential for development of new diagnostic, prognostic, theranostic, and therapeutic tools.
A robust and specific method of miRNA detection and quantification is a critical requirement for profiling miRNA expression. In this study, methods for miRNA detection were compared using myoblast differentiation into myotubes as a model system. Three methods for miRNA detection were compared: the miScript PCR System (QIAGEN), a PCR-based assay from an alternative supplier, and solution hybridization using radiolabeled RNA probes. Using the miScript PCR System, a single RNA sample was reverse transcribed, and the cDNA was used for real-time PCR profiling of multiple miRNAs. Using the alternative PCR-based assay, it was necessary to perform a unique reverse-transcription (RT) reaction for each miRNA to be quantified, which greatly increases the cost and complexity of miRNA analysis. Solution hybridization involved the preparation of specific radiolabeled probes for each assay and a time-consuming solution hybridization step. Unhybridized RNA and probe were removed in an RNase digestion step before samples were analyzed on a polyacrylamide gel.
Often when studying miRNA expression, it is desirable to discriminate between levels of mature miRNAs and processing intermediates. In these cases, the PCR-based assay used should detect only mature miRNAs. To verify that only mature miRNAs were being detected using the miScript PCR System, components of the miRNA processing machinery were knocked down prior to miRNA detection and the results were then compared to control samples.
Materials and methods
Cell differentiation
C2C12 cells were cultured as myoblasts on collagen-coated dishes in DMEM with 10% FBS, and then induced to differentiate into TD myotubes by serum deprivation for 72 hours in the presence of 50 mM 1-D-arabinofuranosylcytosine (1).
miRNA detection
Total RNA from myoblasts and myotubes was used for miRNA detection using the miScript PCR System, an alternative PCR-based assay, or solution hybridization with radiolabeled RNA probes according to the manufacturers’ instructions.
Gene knockdown
Primary mouse embryo fibroblasts (PMEFs) were cultured in DMEM with 10% FBS and infected with a control lentivirus or with lentivirus expressing miR-shRNA for Dicer, DGCR8, and Drosha using pSico-R PGK-puro vector (2). Cells were selected using puromycin and harvested for subsequent RNA purification and mRNA/miRNA detection.
Results
miScript PCR System enables miRNA analysis with less RNA than solution hybridization
Using either the miScript PCR System or solution hybridization, miRNA expression analysis was performed for a range of miRNAs on RNA purified from myoblasts and myotubes. With the miScript PCR System, 1 µg total RNA was sufficient to generate cDNA for 100–500 reactions for reliable detection of miRNAs. In contrast, with solution hybridization, 1–4 µg total RNA was necessary for detection of a single miRNA.
Both solution hybridization (data not shown) and the miScript PCR System identified 2 miRNAs whose steady state expression levels significantly changed after myotube formation. miR-16 had a ~2-fold higher level of expression in myoblasts compared to myotubes, whereas miR-133a had a ~0.5-fold lower level of expression in myoblasts compared to myotubes (see figure
miRNA expression analysis of C2C12 myoblasts and myotubes).
Multiple PCRs from a single RT reaction using the miScript PCR System
Expression of 8 miRNAs was analyzed in myoblasts and myotubes using either the miScript PCR System or a PCR-based assay from an alternative supplier. With the miScript PCR System, one RT reaction was sufficient to profile the expression of hundreds of miRNAs. However, with the alternative PCR-based assay, one RT reaction enabled analysis of just one miRNA. Therefore, the miScript PCR System provided increased flexibility and decreased time and effort for analysis of multiple miRNAs. Of the 8 miRNAs analyzed, 2 showed increased expression in myoblasts (miR-16 and miR-25), 3 showed decreased expression in myoblasts (miR-100, miR-21, and miR-133a), and 3 showed no change in expression between myoblasts and myotubes (miR-30c, miR-17, and let-7b) (see figure
Expression of 8 miRNAs detected using 2 PCR systems).
Detection of mature miRNA only and not precursor miRNA
To ensure reliable interpretation of results, it is important to ensure that the miRNA analysis method used detects mature miRNAs, which are biologically active, and not precursor miRNAs, which have not completed processing to become active molecules. Knockdown of critical components in the miRNA processing pathway was performed to test whether the miScript PCR System detected mature miRNAs only. Dicer, Drosha, and DGCR8 are central to the miRNA processing mechanism (3). Their knockdown prevents accumulation of mature miRNAs in mammalian cells. Using the miScript PCR System and 4 miScript Primer Assays, expression analysis of 4 miRNAs was performed following knockdown of each component and was compared to miRNA expression in control cells. miRNA expression was detected in control cells. In contrast, significantly decreased miRNA expression was detected in cells in which miRNA processing components were knocked down (see figure
Decreased miRNA detection in the absence of mature miRNAs). The expression of miR-16 was higher than the other miRNAs tested. This could indicate that miR-16 stability is higher than the other miRNAs tested, resulting in a higher level of expression over the same time period. These results strongly indicate that the miScript PCR System can be used to detect mature miRNAs only.
Conclusions
- The miScript PCR System enabled reliable, efficient miRNA detection. A benefit of this system was that it required less starting RNA compared to solution hybridization.
- Using the miScript PCR System, hundreds of miRNAs can be detected from a single RT reaction, providing increased flexibility compared to an alternative PCR-based assay.
- Knockdown of miRNA processing genes significantly decreased miRNA detection using the miScript PCR System. This indicates that the miScript PCR System can be used to detect mature miRNAs only.
References
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