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Biochemical Molecules displacement of prepared rooms with professional equipments for RNA detection

Supplementary Information for

“A convenient system for highly specific and sensitive detection of

miRNA expression”


Xiangqi Li, Minjie Ni, Chaobao Zhang, Wubin Ma, Yonglian Zhang



SUPPLEMENTAL DATA

Primer sequences for PCR amplification (shown 5′ to 3′) are as follows:

Primer name

Primer sequence

RmiR326-RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAactgga

RmiR326-RT2

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAactggagg

RmiR326-PCR

TCTTTcctctgggcccttcc

RmiR291-3P-RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAggcaca

RmiR291-3P-RT2

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAggcacaca

RmiR291-3P-PCR

CCCaaagtgcttccactttgt

RmiR295-RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAagaagtg

RmiR295-RT2

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAagaagtgt

RmiR295-PCR

TCCactcaaatgtggggcac

RmiR3085-RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAgagggg

RmiR3085-RT2

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAgagggggc

RmiR3085-PCR

TCTtctggctgctatggcc

RmiR99b * -RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAcggacc

RmiR99b * -RT2

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAcggaccca

RmiR99b * -PCR

CCTcaagctcgtgtctgtgg

RmiR125a-3p-RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAggctcc

RmiR125a-3p-RT2

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAggctccca

RmiR125a-3p-PCR

CTCacaggtgaggttcttgg

RmiR292-5p-RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAcaaaag

RmiR292-5p-RT2

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAcaaaagag

RmiR292-5p-PCR

CTCCactcaaactgggggct

RT-miR-138

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAcggcct

RmiR138-PCR

CGGagctggtgttgtgaatc

RmiR-122 -RT

TGGACGACCGTGTCGTGGAGTCGGCTAATGGTCGTCCAcaaaca

RmiR-122-PCR

GACCtggagtgtgacaatgg



SUPPLEMENTAL FIGURE LEGENDS

Fig. S1 Detection of miRNAs under different conditions by stem-loop PCR.

MiR-122, -138, -99b*, -125a-3p, -291, -292-5p, -295, -326, -3085 are used as examples to illustrate the difficulties involved in designing primers and performing experiments for stem-loop PCR. PCR was performed without template, but with RT primers by setting the annealing temperature at 60°C and running 30 cycles. (A) RT primers bind to the 3′ portion of miRNA molecules with six bases. PCR was performed with Taq DNA polymerase (Takara). (B) RT primers bind to the 3′ portion of miRNA molecules with eight bases. PCR was performed with Taq DNA polymerase (Takara). (C) RT primers bind to the 3′ portion of miRNA molecules with six bases. PCR was performed with Kod-plus DNA polymerase (TOYOBO).


Fig. S2 The sensitivity of the detection of miR-7578 and distinguishing of miR-7578 from its precursor by Northern blotting.

The indicated amounts of oligo DNA of rno-miR-7578 and its precursor were hybridized with 5 pmol of its antisense DNA probe at 42°C using conventional radioisotope-based Northern blotting. The time of autoradiography was 12h. pre-miR-7578 indicates the precursor of rno-miR-7578.


Fig. S3 Detection of miR-29a in different cell lines by different methods.

Total RNAs isolated from JEG-3, MG-63, U-2 OS, 5637, and T24 cells were respectively hybridized with the rno-miR-29a DNA probe using different methods. (A) Using conventional radioisotope-based Northern blotting. (B) Using aLHCD. PCR was performed with 20 cycles. The amount of starting RNA was 20 µg for Northern blotting and 1 µg for aLHCD. The amounts of probe were 5 pmol for Northern blotting and 4 pmol for aLHCD. The hybridization temperature was 42°C for Northern blotting and 45°C for aLHCD. The time of autoradiography was 12h, but color development with BCIP/NBT was 2min.


Fig. S4 Quantitative detection of small RNA levels using U6 as an internal control by LH-PCR.

Artificial small RNA probe (4 pmol) was hybridized with the RNA mixtures at 55°C and U6 probe at 65°C. The RNA mixtures included artificial small RNAs and the RNAs isolated from 293FT cells. I1, 0.5 µg of 293 RNAs + 0.25 ng of artificial small RNA; I2, 1 µg of 293 RNAs + 1 ng of artificial small RNA; I8, 1 µg of 293 RNAs + 4 ng of artificial small RNA. Samples were amplified with U6 primers for determination of the initial relative quantity of RNA in each sample, and then all targeted PCR products were normalized to that amount. U6 probe sequence (shown 5′ to 3′): tggaacgcttcacgaatttgcgtgtcatccttgcgcaggggccatgctaa; U6 bridging primer sequences: AD-U6-F, agatgTGGTACTGATGTGATGGACTtggaacgcttcacgaatttgc; AD-U6-R, tcaccTCATATCACACAGCACCGATttagcatggcccctgcgcaa.

Fig. S5 Evaluation of LH-PCR amplification efficiencies of miR-122 and its precursor.

miR-122 probe (40 fmol) was hybridized with 20 fmol rno-miR-122 or its precursor at 50°C. Q-PCR using SYBR Green was performed to evaluate the relative amplification efficiencies of rno-miR-122 and its precursor.


Fig. S6 Evaluation of LH-PCR amplification efficiencies of miR-200b and its family members.

miR-200b probe (0.4 pmol) was hybridized with 0.2 pmol miR-200b/a/c at 58°C. Q-PCR using SYBR Green was performed to evaluate the relative amplification efficiencies of miR-200b and its family members.


Fig. S7 Detection of miR-29 family members by conventional Northern blotting.

Synthetic miRNAs (0.05 ng) and 10 µg of RNAs extracted from caput epididymis were hybridized with the antisense DNA probes of miR-29a at 65°C using conventional radioisotope-based Northern blotting. The time of autoradiography was 12h.


Fig. S8 Detection of miR-138 and its precursor from tissue samples by stem loop-PCR .

preRNAs (20 fmol) and 1 µg of total RNAs were used as the starting materials. Stem–loop PCR was performed with 30cycles. The samples were miR-138 precursor, HeLa cells, and mouse liver, kidney, lung, and brain. PCR products (8 µl) were run on a 2 % agarose gel.



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