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A rapid method to screen putative mRNA targets of any known microRNA.

Huang Y, Qi Y, Ruan Q, Ma Y, He R, Ji Y, Sun Z - Virol. J. (2011)

Bottom Line: Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro.Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1.Our study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virus Laboratory, the Affiliated Shengjing Hospital, China Medical University, 110004 Shenyang, Liaoning, PR China.

ABSTRACT

Background: microRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression by binding to specific sequences on target mRNAs. However, functional identification of mRNA targets is usually difficult and time consuming. Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro.

Results: Fifteen putative target mRNAs for human cytomegalovirus (HCMV) miR-UL112-1, including previously confirmed HCMV IE72, were identified from mRNA-derived cDNAs using hybrid-PCR. Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1.

Conclusions: Our study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.

Show MeSH
Protocol of hybrid-PCR. (A) Schematic presentation of principle and process designed for hybrid-PCR. (B) Diagram showing sequences of miR-UL112-1 and miR-UL112-1 hybrid primer. Positions marked by Red R meant random insertions of A or G. Seed region was indicated by green box surrounding nucleotide 2-7 of miR-UL112-1.
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Figure 1: Protocol of hybrid-PCR. (A) Schematic presentation of principle and process designed for hybrid-PCR. (B) Diagram showing sequences of miR-UL112-1 and miR-UL112-1 hybrid primer. Positions marked by Red R meant random insertions of A or G. Seed region was indicated by green box surrounding nucleotide 2-7 of miR-UL112-1.

Mentions: miRNAs play the role of posttranscriptional regulation by binding to target mRNAs, hence the target sequences were screened among mRNA-derived cDNAs in hybrid-PCR. An oligo dT-3 sites adaptor primer was introduced into 5'-terminal of mRNA-derived cDNA during reverse transcription (Figure 1A). This primer distinguished the mRNA-derived cDNAs effectively from other DNAs or RNAs in amplification. miRNA specific hybrid-primer was designed according to the miRNA sequence. The reverse and complementary sequence of the seed region of miRNA was lacated at the 3'terminal of the hybrid-primer. Hybrid-PCR was projected as semi-nested PCR using the hybrid-primer and the outer/inner primers homologous to the oligo dT-3 sites adaptor primer. Specificity of target mRNA of a given miRNA was determined by hybridization of the hybrid-primer to the sequence of mRNA-derived cDNA. A low annealing temperature of 37°C was applied in the first round amplification, so as to make hybrid-primer hybridize with putative target sequences in a condition similar to core body temperature. Then a second round PCR with higher annealing temperature of 55°C was followed for further specific amplification of sequences from putative target mRNAs. Extension was long enough to avoid incomplete amplification. The products of amplification were variable in length (Figure 2A).


A rapid method to screen putative mRNA targets of any known microRNA.

Huang Y, Qi Y, Ruan Q, Ma Y, He R, Ji Y, Sun Z - Virol. J. (2011)

Protocol of hybrid-PCR. (A) Schematic presentation of principle and process designed for hybrid-PCR. (B) Diagram showing sequences of miR-UL112-1 and miR-UL112-1 hybrid primer. Positions marked by Red R meant random insertions of A or G. Seed region was indicated by green box surrounding nucleotide 2-7 of miR-UL112-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3025964&req=5

Figure 1: Protocol of hybrid-PCR. (A) Schematic presentation of principle and process designed for hybrid-PCR. (B) Diagram showing sequences of miR-UL112-1 and miR-UL112-1 hybrid primer. Positions marked by Red R meant random insertions of A or G. Seed region was indicated by green box surrounding nucleotide 2-7 of miR-UL112-1.
Mentions: miRNAs play the role of posttranscriptional regulation by binding to target mRNAs, hence the target sequences were screened among mRNA-derived cDNAs in hybrid-PCR. An oligo dT-3 sites adaptor primer was introduced into 5'-terminal of mRNA-derived cDNA during reverse transcription (Figure 1A). This primer distinguished the mRNA-derived cDNAs effectively from other DNAs or RNAs in amplification. miRNA specific hybrid-primer was designed according to the miRNA sequence. The reverse and complementary sequence of the seed region of miRNA was lacated at the 3'terminal of the hybrid-primer. Hybrid-PCR was projected as semi-nested PCR using the hybrid-primer and the outer/inner primers homologous to the oligo dT-3 sites adaptor primer. Specificity of target mRNA of a given miRNA was determined by hybridization of the hybrid-primer to the sequence of mRNA-derived cDNA. A low annealing temperature of 37°C was applied in the first round amplification, so as to make hybrid-primer hybridize with putative target sequences in a condition similar to core body temperature. Then a second round PCR with higher annealing temperature of 55°C was followed for further specific amplification of sequences from putative target mRNAs. Extension was long enough to avoid incomplete amplification. The products of amplification were variable in length (Figure 2A).

Bottom Line: Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro.Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1.Our study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virus Laboratory, the Affiliated Shengjing Hospital, China Medical University, 110004 Shenyang, Liaoning, PR China.

ABSTRACT

Background: microRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression by binding to specific sequences on target mRNAs. However, functional identification of mRNA targets is usually difficult and time consuming. Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro.

Results: Fifteen putative target mRNAs for human cytomegalovirus (HCMV) miR-UL112-1, including previously confirmed HCMV IE72, were identified from mRNA-derived cDNAs using hybrid-PCR. Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1.

Conclusions: Our study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.

Show MeSH