Study on the role of gga-miRNA-200a in regulating cell differentiation and proliferation of chicken breast muscle by targeting Grb2

Figures & data

Table 1. The primers used for PCR or qPCR

Primer name	Primer sequences	TM (°C)	Product
Grb2-qPCR-F	AGGCAAACAGAGACACGATG	56	160bp
Grb2-qPCR-R	GAACTTCACCACCCAGAGGA
pre-miR-200a-F	ccgCTCGAGCCGGTGATGTAAGGCAATCT	59	382bp
pre-miR-200a-R	tgcTCTAGAGGTTTCCAGCAGCTTCTTTG
Grb2-3’UTR-F	ccgCTCGAGTGAAGAATTCAATGTATATT	59	291bp
Grb2-3’UTR-R	atttGCGGCCGCACTTTCTCCCCCTCTCCTTG
Grb2-3’UTR-OF	ATAGGCCTTTTTTATTTAATTTTTTTTTTTAATATATATACAC	59	284bp
Grb2-3’UTR-OR	GTGTATATATATTAAAAAAAAAAATTAAATAAAAAAGGCCTAT
β-actin-F	GAGAGAAGATGACACACAGATC	56	155bp
β-actin-R	GTCCATCACAATACCAGTGG

Note: Restriction sites were underlined, protective bases were dashed. Grb2 3’UTR-OF represents to forward primer for overlap PCR, Grb2 3’UTR-OR represents reverse primer for overlap PCR.

Figure 1. Grb2 relative expression level in breast muscle. Grb2 expression profile in breast muscle of juvenile (20 weeks old) and aged (55 weeks old) chicken.

Note: *** represents for P < 0.001

Figure 2. Prediction of targeting relationship between gga-miR-200a-3p and Grb2. (a) Sequence alignments of gga-miR-200a-3p and the target site in the 3’ UTR of Grb2. (b) Secondary structure of the RNA duplex of gga-miR-200a-3p targeting Grb2 3’ UTR target site respectively (Red: Target sequence; Green: gga-miR-200a-3p).

Figure 3. gga-miR-200a relative expression level in breast muscle. gga-miR-200a expression profile in breast muscle of juvenile (20 weeks old) and aged (55 weeks old) chicken.

Note: ** represents for P < 0.01

Figure 4. Verification of the targeting relation between gga-miR-200a and Grb2. (a) Eukaryotic expression vector and dual fluorescence report vector construction. Sequence alignment of pcDNA3.1-miR-200a recombinant expression vector with miR-200a precursor sequence obtain from NCBI. (b) Sequence alignment of psiCHECK 2-Grb2 3’UTR and psiCHECK 2-Grb2 3’UTR-mut (knocked out the miR-200a binding sites) recombinant expression vector with Grb2 3’UTR sequence obtained from NCBI. (c) DF1 cell before (left) and after (right) transfection of the vectors. (d) Dual-luciferase report assays illuminated gga-miR-200a is the positive target of Grb2 and can suppress the expression level of Grb2.

Note: ** represents for P < 0.01