MicroRNA

Abstract

MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene-expression posttranscriptionally. MiRNA research in allergy is expanding because miRNAs are crucial regulators of gene expression and promising candidates for biomarker development. MiRNA mimics and miRNA inhibitors currently in preclinical development have shown promise as novel therapeutic agents. Multiple technological platforms have been developed for miRNA isolation, miRNA quantitation, miRNA profiling, miRNA target detection, and modulating miRNA levels in vitro and in vivo. Here we will review the major technological platforms with consideration given for the advantages and disadvantages of each platform.

Keywords: Allergy; CRISPR; CRISPR/Cas9; antagomiRs; antimiRs; gene silencing; in situ hybridization; miRNA; miRNA expression; miRNA inhibitors; miRNA isolation; miRNA mimics; miRNA profiling; miRNA quantitation; miRNA targets; microRNA; microRNA methods; microarray; qPCR; stem-loop.

Figure 1

miRNA Extraction methods. (A): Phenol-chloroform based phase separation followed by pelleting of RNA and re-dissolve in nuclease free water. (B): The Aqueous phase from the phenol-chloroform can be loaded onto an RNA adsorbing column. After wash steps, RNA is eluted in nuclease free water. Alternatively, newer miRNA extraction kits allow the user to skip the phase separation step. The samples homogenized in phenol can be loaded directed onto an RNA adsorbing column, followed by wash steps and elution of RNA. A proprietary lysis buffer may be provided by the manufacturer instead of the phenol based reagent.

Figure 2

Quantitative RT-PCR for miRNA detection. (A): Reverse transcription with miRNA specific stem-loop primer, followed by qPCR using a miRNA specific probe, a miRNA specific forward primer (Green) and reverse primer complementary to the stem-loop sequence (Orange). (B) Universal reverse transcription by adding polyA tails to 3′end of miRNA, followed by reverse transcription with a Poly T primer with a universal sequence (Tag) appended at the 3′end, then qPCR with miRNA specific forward primer (Green) and universal reverse primer (Orange). (C) Universal reverse transcription by adding a 5′adapter sequence and polyA tails to 3′end of miRNA, followed by reverse transcription with a Poly T primer with a universal sequence (Tag) appended at the 3′end, followed by optional universal amplification step with 5′ primer binding to the adapter sequence (Blue) and 3′primer binding to the tagged sequence (orange), followed by qPCR with a miRNA specific probe, a miRNA specific forward primer (Green) and a universal reverse primer (Orange).

Figure 2

Quantitative RT-PCR for miRNA detection. (A): Reverse transcription with miRNA specific stem-loop primer, followed by qPCR using a miRNA specific probe, a miRNA specific forward primer (Green) and reverse primer complementary to the stem-loop sequence (Orange). (B) Universal reverse transcription by adding polyA tails to 3′end of miRNA, followed by reverse transcription with a Poly T primer with a universal sequence (Tag) appended at the 3′end, then qPCR with miRNA specific forward primer (Green) and universal reverse primer (Orange). (C) Universal reverse transcription by adding a 5′adapter sequence and polyA tails to 3′end of miRNA, followed by reverse transcription with a Poly T primer with a universal sequence (Tag) appended at the 3′end, followed by optional universal amplification step with 5′ primer binding to the adapter sequence (Blue) and 3′primer binding to the tagged sequence (orange), followed by qPCR with a miRNA specific probe, a miRNA specific forward primer (Green) and a universal reverse primer (Orange).

Figure 2

Quantitative RT-PCR for miRNA detection. (A): Reverse transcription with miRNA specific stem-loop primer, followed by qPCR using a miRNA specific probe, a miRNA specific forward primer (Green) and reverse primer complementary to the stem-loop sequence (Orange). (B) Universal reverse transcription by adding polyA tails to 3′end of miRNA, followed by reverse transcription with a Poly T primer with a universal sequence (Tag) appended at the 3′end, then qPCR with miRNA specific forward primer (Green) and universal reverse primer (Orange). (C) Universal reverse transcription by adding a 5′adapter sequence and polyA tails to 3′end of miRNA, followed by reverse transcription with a Poly T primer with a universal sequence (Tag) appended at the 3′end, followed by optional universal amplification step with 5′ primer binding to the adapter sequence (Blue) and 3′primer binding to the tagged sequence (orange), followed by qPCR with a miRNA specific probe, a miRNA specific forward primer (Green) and a universal reverse primer (Orange).

Figure 3

MiRNA target detection. (A) An illustration of a miRNA/mRNA base pair, highlighting the critical seed region required for miRNA targeting. (B) Target verification by cloning the 3′UTR of the target mRNA into a luciferase reporter vector, followed by co-transfection of miRNA mimics/inhibitors and reading the luciferase activity. (C) Specific miRNAs are targeted by transfecting CRISPR/Cas9 targeting sequence, followed by puromycin selection and single clone isolation. The clone with loss of miRNA expression (Red) is expanded and whole transcriptome sequencing is performed to identify direct and indirect targets. (D). Protein/DNA in cells are UV cross linked, followed by cell lysis and Immunoprecipitation of AGO protein in the RNA induced silencing complex. A 3′ linker is ligated, the RNA induced silencing complex is digested by proteinase K and a 5′ linker is ligated. This is followed by reverse transcription, PCR amplification and high-throughput sequencing.

Figure 3

MiRNA target detection. (A) An illustration of a miRNA/mRNA base pair, highlighting the critical seed region required for miRNA targeting. (B) Target verification by cloning the 3′UTR of the target mRNA into a luciferase reporter vector, followed by co-transfection of miRNA mimics/inhibitors and reading the luciferase activity. (C) Specific miRNAs are targeted by transfecting CRISPR/Cas9 targeting sequence, followed by puromycin selection and single clone isolation. The clone with loss of miRNA expression (Red) is expanded and whole transcriptome sequencing is performed to identify direct and indirect targets. (D). Protein/DNA in cells are UV cross linked, followed by cell lysis and Immunoprecipitation of AGO protein in the RNA induced silencing complex. A 3′ linker is ligated, the RNA induced silencing complex is digested by proteinase K and a 5′ linker is ligated. This is followed by reverse transcription, PCR amplification and high-throughput sequencing.

Figure 3

MiRNA target detection. (A) An illustration of a miRNA/mRNA base pair, highlighting the critical seed region required for miRNA targeting. (B) Target verification by cloning the 3′UTR of the target mRNA into a luciferase reporter vector, followed by co-transfection of miRNA mimics/inhibitors and reading the luciferase activity. (C) Specific miRNAs are targeted by transfecting CRISPR/Cas9 targeting sequence, followed by puromycin selection and single clone isolation. The clone with loss of miRNA expression (Red) is expanded and whole transcriptome sequencing is performed to identify direct and indirect targets. (D). Protein/DNA in cells are UV cross linked, followed by cell lysis and Immunoprecipitation of AGO protein in the RNA induced silencing complex. A 3′ linker is ligated, the RNA induced silencing complex is digested by proteinase K and a 5′ linker is ligated. This is followed by reverse transcription, PCR amplification and high-throughput sequencing.