In this study, we collected and processed four human prostate cancer associated miRNA microarray expression datasets with newly developed cancer outlier detection methods to identify differentially expressed miRNAs DE-miRNAs. The targets of these DE-miRNAs were then extracted from database or predicted by bioinformatics prediction and then mapped to functional databases for enrichment analysis and overlapping comparison.
Newly developed outlier detection methods were found to be more appropriate than t-test in cancer research, and the consistency of independent prostate cancer expression profiles at pathway or gene-set level was shown higher than that at gene i. Among the top 15 GeneGO pathways, 5 were reported previously and the rest could be putative ones. Our analyses showed that more appropriate outlier detection methods should be used to detect oncogenes or oncomiRNAs that are altered only in a subset of samples.
We also found that the utilization of similar meta-analysis methods between miRNA and mRNA profiling datasets result in the detection of the same pathways.
micro RNA and qRT-PCR
Plant material and phloem sap collection Arabidopsis thaliana Columbia, Cucurbita maxima 'Crown' pumpkin and Cucumis sativus 'Telegraf' cucumber plants were grown in a greenhouse under natural daylight conditions. UPL probe 21 was obtained from universal probe library database Roche Diagnostics. Stem-loop RT primers were designed according to Chen et al. Sequence data are presented in Table 1.
Table 1 miRNA, primer and probe sequences. UPL probe 21 reverse complement sequence is highlighted in bold. Equipment Standard laboratory equipment including a thermal cycler is required for pulsed reverse transcription and end-point PCRs. Stem-loop pulsed reverse transcription protocol 1. For a 'no RNA' master mix, add the following components to a nuclease-free microcentrifuge tube: 0.
For a 'no RT primer' master mix, add the following components to a nuclease-free microcentrifuge tube: 0. Assemble the RT reaction. Prepare a PCR master mix by scaling the volumes listed below to the desired number of amplification reactions. Also prepare water controls by adding nuclease-free water in place of the RT product. Add the following components to a nuclease-free microcentrifuge tube: Seal each capillary with a stopper.
Analyse results using the LightCycler software. Perform a Relative Quantification-Monocolor Analysis. Analyse results using the LightCycler software as described above. This amplification was performed in a semi-quantitative manner, using 20—35 cycles Figure 2B. Similarly, no amplification was obtained from the water control data not shown.
However, 40 or more cycles of PCR often gave rise to some non-specific amplification in control reactions data not shown. Therefore, all further end-point amplification reactions were limited to 35 cycles. RNA gel blot analyses clearly show higher expression of pri-miR in the shoot tip and stem than in leaf tissue, and higher expression of mature miRNA in leaf than in shoot tip and stem. Similar results were obtained with cucumber tissue RNA data not shown.
We further analysed the expression of several miRNAs in pumpkin tissues. RNA gel blot analysis suggested higher expression in leaf than in the shoot apex or stem tissue for miR; highest expression in the shoot apex, followed by leaf and low expression in the stem tissue for miR; miR and miR showed similar levels of expression across all analysed tissues Figure 4A.
Figure 4 Detection of miRNAs in tissue and phloem sap.
Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs
We were again able to detect miR, miR and miR from as little as 20 pg total RNA without significant amplification in minus-RT control and the water control. However, a larger number of amplification cycles often resulted in non-specific amplification in the 40—80 bp range that was difficult to distinguish from the desired amplification product. These non-specific products were found in the minus-RT and water controls and were often indistinguishable from the specific PCR products by melting-curve analysis. Melting-curve analysis could not distinguish between the specific and non-specific PCR products, but cloning and sequencing of amplicons derived from minus-RT control revealed that they were concatenated primer sequences data not shown.
This assay established that the phloem sap is enriched for miR and miR; phloem sap showed a fold increase in miR expression and a fold increase in miR expression compared with the surrounding vasculature. The abundance of miRNA was similar in the phloem sap and in the surrounding vascular tissue. Only relatively low levels of miR expression were detected in the vascular tissue, but very little or no expression was detectable in the phloem sap greater than fold below the levels detected in the vasculature; Figure 7A. The results were comparable to those obtained by RNA gel blot analyses Figure 7B and spatial analysis using in situ hybridization [ 35 ] that revealed high expression of several miRNA, including miR, but not miR, in the vascular bundles of Arabidopsis thaliana and Nicotiana benthamiana.
Figure 7 Expression of miRNAs in vascular tissue and phloem sap. Plant Physiol. Nat Genet. J Cell Physiol. Plant Cell. Plant J. Indirect detection methods primarily include Northern blotting, microarray, and reverse transcription-polymerase chain reaction RT-PCR. Although widely used, both the Northern blotting and microarray methods are semiquantitative and suffer from poor sensitivity and they require large amounts of the starting RNA. Although microarrays offer high-throughput detection of miRNAs and the potential capability of absolute quantification Bissels et al.
Recent attempts to measure miRNAs with isothermal methods have met with some success, but are labor intensive Cheng et al. However, due to an additional probe hydrolysis step, TaqMan assays were not compatible with fast thermocycling protocols for rapid detection of miRNAs.
Furthermore, with the escalating identification of hundreds of candidate miRNAs by deep sequencing Bar et al. Attempts have been made to improve miRNA detection without reliance on fluorescent probes Raymond et al. It has been noticed that a consensus strategy of these assays and some other TaqMan assays Varkonyi-Gasic et al.
Furthermore, it is yet to be determined whether these assays are capable of rapid, multiplexed, and direct detection of miRNAs without RNA isolation. In this study, we present a novel approach by coupling a deoxyuridine-incorporated RT oligonucleotide with a secondary structure and a hemi-nested real-time PCR for the rapid and robust quantification of mature miRNAs directly from cultured cells. This miRNA assay showed a high dynamic range, efficiency, and sensitivity of detection and excellent discrimination of the target mature miRNA against its precursor form and homologous family members.
Instead of using a fluorescent probe, we found that a hemi-nested reverse PCR primer could drastically enhance the assay specificity and allow quantification of subzeptomole amounts of miRNA using SYBR Green I under rapid thermocycling conditions. Furthermore, we showed that miRNAs can be efficiently quantified directly from ten to cells using this multiplexed assay without laborious RNA isolation. Initially, we noticed that in a number of previous reports Chen et al. To achieve specificity, a unique miRNA-specific fluorescent probe is required to discriminate the targets from the nonspecific amplicons Chen et al.
In the case of some TaqMan-based assays, this specificity was compromised for the convenience of throughput by using one common probe for detection. We hypothesized that increased specificity can be achieved by designing a hemi-nested reverse PCR primer instead of using a common or universal reverse PCR primer. The cDNA sample was then amplified using a tagged forward primer Pf and a hemi-nested reverse primer Pr , where 3—5 nt extend beyond the RT oligonucleotide.
Pf, forward primer; Pr, reverse primer; dU, deoxyuridine. The assay exhibited excellent dynamic range and linearity under both cycling profiles Fig. The excellent linearity of the standard curve suggested that the miR assay had a wide dynamic range of at least 7 logs and was able to detect as few as copies subzeptomoles per RT reaction Fig.
Standard curves are plotted as Ct versus Log starting material per RT. Total RNA dilutions from ng to 1 pg Fig. The standard curves again showed excellent linearity Fig. Here, ng of total RNA from U cells, which did not contain let-7d and let-7e data not shown , was spiked with varying amounts of synthetic let-7d or let-7e miRNAs. Furthermore, these assays showed specific amplifications of target miRNAs from both synthetic standards and total RNA samples. The sizes of the amplicons were verified by gel electrophoresis Supplemental Fig.
Several miRNA families e. The eight let-7 family miRNAs share up to Each assay was used to amplify all eight synthetic let-7 miRNAs and the relative detection was compared. For let-7 miRNAs that differ by 2 nt or more, these assays were able to specifically detect the target miRNA with less than 0.
Discrimination of human let-7 homologs.
A Sequence alignment of the eight let-7 family miRNAs. Discrimination between certain highly homologous miRNAs can be further improved using a novel strategy involving a UDG treatment. In this study, we observed that the amount of RT oligonucleotides carried over was able to serve as amplification primers during PCR, although with poorer efficiency Supplemental Fig. We then hypothesized that discrimination between let-7 miRNA homologs may be further improved with a similar strategy. Indeed, while the standard dT RT oligonucleotide for let-7a showed a relative detection of 0.
Both the let-7a dT and dU RT oligonucleotides were able to prime reverse transcription of let-7a equally well data not shown. The dU residues are underlined. B Relative detection of the let-7f miRNA by the let-7a assay. Copies 10 9 of let-7a or let-7f were reverse transcribed using or nM of the dT, dU1, or dU2 RT oligonucleotide. Nonspecific amplification of the let-7f miRNA was calculated and expressed as a percentage of relative detection. Error bars indicate standard deviations of quadruplicate measurements. A total of 26 miRNAs were examined including 18 miRNAs miR-7, b, b, , , , , , b, a, b, c, , , , , , and reported to be dysregulated in the human glioblastoma Pang et al.
Twenty miRNAs except for miR, b, c, , let-7d, and let-7e were found to be expressed in U cells data not shown. No significant regulation of the other miRNAs was observed. Error bars indicate standard deviations of triplicate measurements. We then developed a multiplexed assay for simultaneous reverse transcription of the 26 miRNAs corresponding to 24 RT oligonucleotides and subsequent detection of individual miRNAs by hemi-nested real-time PCR. A similar result was obtained by a standard single-plex assay using miR and let-7i as examples Supplemental Fig.
Regulation of these miRNAs was expressed as fold changes to nonstimulated control samples. Direct detection of miRNAs from cell lysates can avoid the time-consuming multistep RNA isolation process and dramatically increase the throughput of the assay. We then further investigated the capability of the hemi-nested real-time RT-PCR assay in the direct and multiplex quantification of miRNAs from 10 to , cultured U cells in 96 wells.
The isolated total RNAs from these cells were used as controls for quantification. Importantly, both miR and miR can be detected equally well from the lysates of 10— cells using this multiplex assay, as compared with the isolated RNA controls. However, it is worthy to note that the direct quantification of miRNAs was compromised with the lysates from near-confluent 10 4 cells per well or over-confluent 10 5 cells per well cell densities. Interestingly, no significant difference was observed in the detection of miRNAs from the cell lysates with or without the RNase inhibitors, suggestive of the remarkable stability of mature miRNAs.
Direct and multiplexed detection of miRNAs from cell lysates. U cells cultured in wells at various densities 10, , , 10, and , cells per well were directly lysed and reverse transcribed in a reaction mixture containing 24 miRNA RT oligonucleotides. The cDNA samples 2. With the increased interest in the expression profiles of miRNAs, rapid, robust, and cost-effective methods for the detection of mature miRNAs are highly desirable. The hemi-nested real-time RT-PCR assays described herein were simple to design, showed excellent performance, and provided the flexibility for the design of any miRNAs that may be identified in the future.
In contrast to the previously reported miRNA real-time PCR assays that were performed under standard thermocycling profiles of 45—75 sec per cycle Chen et al.
MicroRNA Expression Detection Methods
Specific and sensitive quantification of mature miRNA from total RNA samples usually requires size fractionation and preamplification, respectively. It has been reported that besides additional sample handling steps, size-fractionation can result in a consistent loss of miRNAs Wang et al. With our miRNA quantitative assay, small amounts of the total RNA from 1 pg to ng and the total RNA isolated from as few as 10 cells can be efficiently detected without the need for fractionation or preamplification.