MicroRNA Experimental Protocol

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Overview

MicroRNAss (miRNAs) are non coding small RNAs, suppressing gene expression or inhibiting translation by binding (sometimes partially) complementary sequences in the 3 UTR of mRNAs. MicroRNAs regulate biological processes including cell proliferation, apoptosis, differentiation, metabolism, development and neoplasmic transformation, among others. MiRNA genes are encoded in intergenic chromosomal regions or within the introns of protein coding genes. MicroRNA genes are transcribed by RNA polymerase II or III, generating RNA molecules containing the characteristic stem loop structures (primary RNA). Drosha-DGCR8 microprocessor complex cuts the primary RNA into shorter ∼70 nt pre-miRNAs. The pre-miRNAs are exported and further processed by an RNase Dicer to imperfectly paired ~22-bp double-stranded mature miRNA. One of the strands is integrated into RNA-induced silencing complex, and functions during post-transcriptional gene regulation.

In miRBase database version 19, released in August 2012, 21264 hairpins and 25141 mature products are listed for 193 species, including 1667 hairpins and 2062 mature products for human. More miRNAs are likely to be found. Perhaps 90% human genes are regulated by microRNAs. However, the regulation on target genes is hard to define and elucidate. Bio-informatics software prediction, integrated with gene chips and biological experiments, have been used to identify the miRNA functions and the taget genes. Gene chip technology profiles the experession of a large number of miRNAs. Comparison of microRNA expression profiles between, for example, normal and tumor tissue samples, can be used for the identification of tumor biomarkers, and even clinial diagnosis of tumors. However, the genechip technology remains semi-quantitative, and needs to be corroborated by other experimental methods. In addition, protein mass spectrometry has been used to identify target genes.

Experimental Authentication of miRNAs

Three methods have been used to detect specific miRNA in tissues or cells: northern hybridization, in situ hybridization, and step-loop real-time PCR. Each has its own advantages and disadvantages. The three methods can be combined to detectad confirm miRNA expression level.

Northern blot

Northern blot is one of widely used methods for detecting RNA levels. MicroRNAs are small molecules and some of microRNA expression levels are very low. The RT-PCR method tends to result in poor reproducibility, and the steps involved are complicated. Most researchers currently use Northern Blot, which has good reproducibility, high sensitivity, a direct approach, and can be used to detect the change in microRNA expression level. Among various ways to detect the hybridized probe, isotope labeling of the probe has some limitations due to concern of radiation contamination. Locked-nucleic acid (LNA) probes, with high stability, specificity, and no radioactive contamination are widely used [1] [2] [3] .

MicroRNA Experimental Protocol Figure 1
Figure 1. Northern blot indicates both pre- and mature miR-499 bands from human and mouse. From [6] .

Basic principles: RNA molecules are denatured and separated by urea polyacrylamide gel electrophoresis, transferred to nylon membrane, fixed, and then hybridized with DNA or RNA probes labelled with an isotope, digoxin, or other markers. The target miRNAs with sequnces complementary with the probe, will be detected and its relative size and intensity can be measured.

  1. prepare 15% of the Urea-PAGE gel without APS and TEMED. 50ml of Pre-Gels: Urea: 24 g; 40% acrylamide: 18.75 mL; 5x TBE Buffer: 10 mL; ddH2O: 0 mL.
  2. clean containers and other devices: containers with western blot device can be used.
    1. wash with water
    2. soak electrophoresis tank, glass, and comb in H2O2 for 10 minutes
    3. wash with water treated with 0.1% DEPC
  3. make gel: Add 33.3- 45 ul APS and 10-20ul TEMED in 10ml pre gel and mix gently, then add it into the glass and insert the comb into the gel.
  4. prepare RNA samples: add 2ul 10xRNA loading buffer in the RNA sample dissolved in the 18ul DEPC H2O. 95 C for 5min, cooled on ice. Samples collected by centrifugation.
  5. run electrophoresis: in 1x TBE. 180V.
    Ambion: 300V pre-electrophoresis 10min (to prevent leakage while activated adhesive glue) before adding the sample, then rinse the holes with electrophoresis solution 1xTBE and carefully add the sample after the urea out of holes.
    Roche: provided LNA-labelled DNA as control, while its synthetic RNA Oligo as positive control (1 pmol total).
    Bromophenol Blue indicates 13nt, Xylene Cyanole FF 40nt.
  6. transfer to membrane: in 0.5x TBE
    1. soak the gel in 0.5x TBE about 10 min.
    2. stain the gel with EtBr (0.5ug/ml) for 10min. observe the RNA by UV. wash the gel with 0.5x TBE for 5 min.
    3. soak the Nylon membrane (GE Corporation) and two pieces of thick filter paper in 0.5x TBE for 10 min.
    4. the filter paper, plastic, film, filter paper form a sandwich structure, the gel should be close to the cathode side. No air bubbles between each layer.
    5. assemble the membrane device
    6. transfer film on ice 1 h 300mA
    7. the membrane (RNA side up) on the UV cross-linking apparatus for use 4000J UV cross-linking
    8. the film clip in the middle of thick filter paper, 80 C baking 0.5 - 2 h.
    9. methylene blue staining 3 ~ 5min.
  7. hybridize
    1. pre-hybridize 1h in in hybridization solution (7% SDS, 0.2M Na2HPO4) at hybridization oven of 37 - 42 C. Add the the membrane into the hybridization bag, and then add the Dig-Labeled probe in the bag, hybridized overnight at the hybridization oven.
    2. 37 - 42 C membrane washing solution (2× SSC, 0.1% SDS), wash membrane three times every 15 min.
    3. room temperature MABT (0.1M Maleic Acid, 0.15M Nacl, 0.3% Tween-20, PH 7.5), wash membrane three times, each time 5 min.
    4. block it with blocking solution for 1h.
    5. add the anti-Dig antibody and incubate it at RT for 40 min. The antibody should be centrifugalized at 12000g for 5min prior to use.
    6. wash membrane with MABT buffer twice, each time 15 min.
    7. balance the membrane in the detection buffer for 5 min.
    8. add 1ml CSPD buffer in the bag, and incubate it for 5 min.
    9. squeeze out the CSPD buffer, and incubate it for 3-15 min at 37℃C
    10. expose for 2 min ~ 1h

notes:

  1. urea solution can not be heated; 15% of the urea-PAGE gel without the APS and TEMED can be stored at room temperature or 4 C refrigerator for a month or so.
  2. all devices should be maintained as RNAase free.
  3. LNA probes should be aliquoted into small packaging before use, and then kept frozen at -70 C refrigerator.
  4. the hybridizations time and temperature need to be optimized with different miRNA.
  5. nylon membrane after UV cross-linking and baking can be stored at 4 C refrigerator for more than six months.
in-situ hybridization

Many protocols and variations for in situ hybridization with different types of the tissues (whole-mount, paraffin-embed or frozon tissues), and with different detection paradigms are available from different sources, especially online.

MicroRNA Experimental Protocol Figure 2
Figure 2. In situ hybridization of miRNA. Both negative and positive controls are important in in situ hybridization. From [7] .

One of widely cited protocols is by Dr. Pena in 2009 [4] , for mammalian tissues fixed with formaldehyde and 1–ethyl–3–(3–dimethylaminopropyl) carbodiimide (EDC). The supplement in that article lists detailed steps of the experimental procedure. Since miRNAs are very small, one of the crucial steps is to prevent the loss of miRNAs during the hybridization procedure. Fixation with EDC can irreversibly immobilize miRNAs at its 5 phosphate to the protein matrix. Fixation with formaldehyde or EDC alone can not prevent the loss of miRNA from tissue sections during in situ hybridization. In addition, the melting temperature between the probe and intended miRNA differs from the usual calculation due to the interference of formamide during the hybridization step. This alteration of melting temperature should be taken into consideration.

stem-loop realtime PCR

Traditional real-time quantitative polymerase chain reaction (PCR) can only be used to detect miRNA precursors, while stem loop real-time quantitative RT PCR technology can be used to detect mature microRNA [5] . Stem loop real-time quantitative RT polymerase chain reaction (PCR) is highly sensitivity and highly specific for the detection of miRNA expression. It includes two steps: design of reverse transcriptase primers with stem-loop structure and RT-PCR with fluorescently labeled miRNA probes. This technology has the following advantages: highly specific; able to distinguish homologous miRNA sequences; broad linear detection range of miRNA concentration; highly sensitive; small sample consumption, only 1 ~ 10 ng of total RNA; can be used with total RNA samples, purified RNA samples, or cell lysates.

Basic principle: microRNA RT-PCR generally uses stem loop structure primers. Such stem loop microRNA probes are designed to detect the targeted miRNAs, and thus possess specific sequences. Small RNA U6 are usually used as internal control, and random primers are used for the reverse transcription of small RNA U6. The reversely transcribed cDNA from RNA samples or cell lysates serve as the template for RT-PCR. Specifically designed forward and reverse primers aginst target miRNA are used to detect specific miRNA, while forward and reverse primers against small RNA U6 are used as the internal control.

Procedure

  1. total RNA extraction with Trizol. same as a regular RNA Trizol extraction. except during isopropanol processing, the sample is chilled at 4 degree overnight. store the extracted RNA in -80 degrees.
  2. reverse transcription of the RNA. use the 12.5 ul system. RNase free water ; Total RNA: 0.625ug; Impron buffer: 2.5ul; dNTPs: 2.5ul; RNase inhibitor: 0.625ul; Impron Mgcl2: 1.5ul; Primer: 0.5ul; Reverse transcriptase: 0.625ul. detailed steps: calculate the amounts of template and RNase free water; add that amounts to PCR tubes; store the left template at -80 C; prepare the reaction mix as above; aliquote the reaction mix to each PCR tubes; and run the reverse transcription reaction. reverse transcription process: 25C 5 min, 42C 60 min, 70C 15 min. and store at 4C.
  3. run the RT-PCR reaction in triplicates. use the following amount in each RT-PCR reaction. SYBR: 5.0ul; Primer mix: 0.2ul; cDNA template: 1.0ul; Water: 3.8ul.
  4. add 3.4 ul cDNA to the sample tubes.
  5. calculate the amounts of SYBR, primer mix and water; prepare the mix; add the mix to the sample tubes;
  6. aspirate 10 ul from sample tubes, and add it to the 96 well plate. in triplicates.
  7. cover the plate, and seal it.
  8. Load the sample to a machine. Specific PCR program is as follows:
    1. 50C 2 min ;
    2. 95C 5 min ;
    3. 95C 30 s ;
    4. 60C 40 s ;
    5. 72C 30 s ;
    6. 95C 15 s ; 60C 30s ;95C 15 s

note:

  1. due to the high sensitiviy of RT-PCR, run experiments in triplicates
  2. the aliquote and sampling amount must be accurate
  3. mix the samples well before aliquoting them to the 96-well plate. The sample tubes can be vortexed (with slow speed to prevent spilling)
MiRNA Target Gene Identification

Bioinformatic and experimental methods have been used to identify the target miRNA genes. In combination, the accuracy can be improved significantly.

Bioinformatics methods use specific algorithms to screen and rank target genes. There are a number of algorithms. Although various prediction methods employ different calculations, they all center on some common features between microRNAs and their target genes.

  1. complementarity between miRNAs and their target sites
  2. conservation of homologous miRNAs between species
  3. thermal stability between miRNA-mRNA double-strand
  4. lack of complex secondary structure at the binding site of microRNAs
  5. miRNA 5 end can bind to the target genes stronger than the 3 end

Very often, different prediction methods are used to generate lists of potential target genes, and the common genes among the lists become the focus of further investigation.

There are three commonly used prediction methods:

  1. miRanda
  2. TargetScan
  3. PicTar
Validation of miRNA Target Genes

Although bioinformatic approach can predict the target genes for microRNA, the predictions need to be validated through experimental approaches. The target genes can be examined on both protein and RNA levels. On the RNA level, the expression levels of many genes can be evaluated through microarray analysis after exogenous expression of specific miRNA in cells.

The most straightforward methods for target gene validation are through quantitative PCR and western blot to examine the RNA and protein levels of specific genes after the transfection or knockdown of specific microRNA.

Related Links

Labome.ORG lists some of the microRNA researchers.

More about microRNA and other microRNA web resources.

microRNA precursor clones

Precursor clones for all human, mouse, and rat microRNAs. Samples: MIR21 precursor clone MIR155 precursor clone MIR146A precursor clone MIR34A precursor clone MIR221 precursor clone MIR17 precursor clone MIR222 precursor clone MIR122 precursor clone MIR16-1 precursor clone MIRLET7A1 precursor clone

microRNA inhibitor clones

Inhibitor clones for all human, mouse, and rat microRNAs. Samples: MIR21 inhibitor clone MIR155 inhibitor clone MIR146A inhibitor clone MIR34A inhibitor clone MIR221 inhibitor clone MIR17 inhibitor clone MIR222 inhibitor clone MIR122 inhibitor clone MIR16-1 inhibitor clone MIRLET7A1 inhibitor clone

microRNA synthetic inhibitors

Synthetic inhibitors for all human, mouse, and rat microRNAs. Samples: MIR21 synthetic inhibitor MIR155 synthetic inhibitor MIR146A synthetic inhibitor MIR34A synthetic inhibitor MIR221 synthetic inhibitor MIR17 synthetic inhibitor MIR222 synthetic inhibitor MIR122 synthetic inhibitor MIR16-1 synthetic inhibitor

References
  1. Kauppinen S, Hornyik C. Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res. 2004;32:e175 PMID 15598818
  2. Várallyay E, Burgyan J, Havelda Z. MicroRNA detection by northern blotting using locked nucleic acid probes. Nat Protoc. 2008;3:190-6 PMID 18274520
  3. Kim S, Li Z, Moore P, Monaghan A, Chang Y, Nichols M, et al. A sensitive non-radioactive northern blot method to detect small RNAs. Nucleic Acids Res. 2010;38:e98 PMID 20081203
  4. Pena J, Sohn-Lee C, Rouhanifard S, Ludwig J, Hafner M, Mihailovic A, et al. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods. 2009;6:139-41 PMID 19137005
  5. Chen C, Ridzon D, Broomer A, Zhou Z, Lee D, Nguyen J, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005;33:e179 PMID 16314309
  6. Shieh J, Huang Y, Gilmore J, Srivastava D. Elevated miR-499 levels blunt the cardiac stress response. PLoS ONE. 2011;6:e19481 PMID 21573063
  7. Barrey E, Saint-Auret G, Bonnamy B, Damas D, Boyer O, Gidrol X. Pre-microRNA and mature microRNA in human mitochondria. PLoS ONE. 2011;6:e20220 PMID 21637849
last modified : 2012-10-21