Authors: Qing ‘En Lim, Guoqiang Wan, Yoon Khei Ho & Heng-Phon Too
This protocol describes a method that guides researchers in designing assays for any miRNA of interest, andalso allows researchers to easily recover and quantify miRNAs without reliance on expensive proprietary kits. Using our novel assay design, we have previously shown that it is possible to effectively discriminate mature miRNAs from their precursors and almost identical homologs in multiplex. We here extend the approach to include very recently sequenced miRNAs for which no commercial assays are yet available yet. We further show that efficient lysis, recovery and detection of miRNAs are achievable with common reagents and basic enzymatic kits.
miRNAs are small RNAs ~18-22 nucleotides long that are important regulators of physiological and disease processes (1-3). The short length of miRNAs presents challenges for quantification by traditional RNA detection methods. Techniques such as Northern blotting (4), microarrays (5,6), and RT-qPCR (7-9) have been modified in order to specifically detect and quantify miRNAs. RT-qPCR remains the most reliable, sensitive and scalable technology for miRNA quantification (10). Nearly all methods require pure RNA from experimental samples as input for reliable results. At the same time, NGS has resulted in accelerated discovery of many novel miRNAs. In this light, an approach that allows assay customization, multiplexed detection of both annotated and novel miRNAs with minimum sample processing is highly desirable.
Here, we describe a protocol for efficient lysis, DNase treatment and direct, multiplexed reverse transcription of cultured cells, using a reaction buffer containing off-the-shelf surfactants that are commonly used in cell lysis (8). Using this protocol, we profiled the expressions of both annotated (miRBase release 15) and novel miRNAs rapidly and reliably from 10 to 10,000 cells and 10 different cell lines without the need for RNA isolation. This cost-effective, robust and reliable protocol for multiplexed detection of miRNAs increases the throughput of the assays enormously.
An overview of experimental design is provided in Figure 1.
Figure 1: Overview of experimental design
SMRT-qPCR Assay Design
The assay design process has been described (8) and is summarized in Figure 2a. Briefly, the mature miRNA sequence(s) of interest is retrieved from miRBase (http://www.mirbase.org/, current release 15). The reverse transcription (RT) primer is designed such that at the temperature used for reverse transcription it adopts a stable stem-loop structure (dG < -1.5), while under qPCR conditions the stem-loop structure is no longer favored (dG > -0.5). mFold software10 (http://mfold.bioinfo.rpi.edu/) is used to predict conformations and free energy values of the reverse transcription primer under the conditions used for reverse transcription and qPCR respectively (Fig. 2b). The last 6 nucleotides at the 3’ end of the reverse transcription primer are the reverse complement of the last 6 nucleotides of the miRNA of interest.
A pair of qPCR primers is then designed where the reverse primer (with respect to the original miRNA sequence) extends at least one nucleotide beyond the 6 nucleotides used for reverse transcription priming while the 5’ end anneals to the stem-loop region of the RT primer (Fig. 2a). The forward primer extends such that it is at most head-to-head with the reverse primer (i.e. PCR gap = 0 nt) and will usually requires a 5’ GC rich sequence for efficient amplification. Due to the short lengths of the miRNAs, qPCR amplicons generated may be of almost indistinguishable size compared to primer-dimer products, unless dissociations are carried out using high-resolution melt analyses. It is thus critical to minimize primer-primer hybridization to avoid non-specific amplification. Most primer designing softwares (e.g. Beacon Designer, DNAman, Premier Primer) allow the evaluation of primer-primer complementarity. There is also a free online edition of Beacon Designer (http://www.premierbiosoft.com/qOligo/Sequence.jsp?PID=1) that is adequate for this purpose (Fig. 2c).
Figure 2: Overview of Assay Design
(a) Schematic view of SMRT-qPCR. The RT primer should adopt a stem-loop structure under RT conditions and unfold under qPCR conditions. (b) Sample result from mfold algorithm when applied to the miR-21 RT primer. (c) Sample result from Beacon Designer algorithm for primer-primer complementarity.
Cells can be lysed in a 96-well format to minimize reagents requirements. Typically, 10,000 cells can be seeded per well without being over-confluent or forming multiple-layers.
Reverse Transcription (Timing: 35 min)
Quantitative PCR (Timing : ~90 min depending on thermal cycler)
3 hours from cell lysis to data analysis
Please refer to Table 5 for troubleshooting procedures.
For steps 2 through 15 we recommend the use of nuclease-free filtered pipettor tips to avoid contamination. Nuclease-free reagents and labware are critical to obtaining reproducible results.
Step 4 : Ensure complete lysis of cells by inspecting wells under the microscope.
We have successfully lysed up to 10,000 cells by incorporating a DNase step as described above before reverse transcription. The method was able to provide comparable or better detection of a panel of miRNAs when compared to total RNA isolation (Figure 3). Notably, this panel included miR-4286, for which no commercial assay is currently available.
Figure 3: Comparison of Direct Lysis RT-qPCR to RNA Isolation
10,000 U251 cells were subjected to the protocol described. Direct lysis resulted in comparable or superior detection of 9 miRNAs including miR-4286 for which no commercial assays are currently available. Ct shown are from biological triplicates.
We are grateful to the Department of Biochemistry for a conducive, collaborative work environment.
Tables: Combined file with all the tables referenced in this protocol.
Table 1: Lysis Buffer Composition
Table 2: RT Master Mix
Table 3: qPCR Master Mix
Table 4: qPCR Thermal Cycling Parameters
Table 5: Troubleshooting Table
Qing ‘En Lim & Yoon Khei Ho, National University of Singapore
Guoqiang Wan & Heng-Phon Too, National University of Singapore, Singapore-MIT alliance
Correspondence to: Heng-Phon Too ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.202. Originally published online 4 January 2011.