DescriptionThe microRNA Marker is a set of three synthetic single-stranded RNA oligonucleotides 17, 21 and 25 residues long that have free 5´ ends (i.e., no 5´ phosphate groups). These oligonucleotides can be used as size markers on denaturing polyacrylamide gels and Northern blots. The microRNA Marker is best visualized by staining with SYBR-Gold instead of ethidium bromide (Figure 1).
The three marker oligos contain the same core sequence so they can be detected by hybridization with the same probe. The sequences of the microRNA marker band are as follows:
25-mer: 5´AGAGCAGUGGCUGGUUGAGAUUUAA 3´
21-mer: 5´AGCAGUGGCUGGUUGAGAUUU 3´
17-mer: 5´CAGUGGCUGGUUGAGAU 3´
Note: The sequence in bold is common to all three oligos.
A 21-mer DNA oligonucleotide complementary to the marker sequences is included. This oligonucleotide is biotinylated at the 3´ end and has a free 5´ end so it can also be labeled with γ-32P-ATP and T4 Polynucleotide Kinase (NEB# M0201). The sequence of the oligonucleotide probe is as follows:
Properties and Usage
- The microRNA Marker is provided in a ready-to-load denaturing solution. Denature by heating for 3-5 minutes at 95°C and place on ice. Load 5-10 µl for staining with SYBR Gold in denaturing gels. In Northern blots, less than 1 µl (12 ng) is sufficient for detection by hybridization.
The Orange G loading buffer migrates faster than the smallest band, and migrates approximately as far as the nucleotides.
- Oligonucleotide Probe Usage: The provided biotinylated probe DNA oligonucleotide can be labeled with T4 Polynucleotide Kinase (NEB #M0201) and radioactive γ-32P-ATP using the following protocol:
1. Mix the following components in a sterile microfuge tube:
Oligonucleotide Probe -- 1-5 µl
10X T4 Polynucleotide Kinase reaction Buffer -- 2.0 µl
γ-32P-ATP (5 µCi/µl) -- 1-2 µl
T4 Polynuclotide Kinase -- 1 µl
Sterile dH20 -- X µl
Total volume -- 20 µl
2. Incubate for 30 minutes at 37°C.
3. Purify labeled probe using a G-25 spin column.
- Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual (3rd Ed.). 7.1-7.56.
- Can I use the microRNA Marker on a non-denaturing gel?
- Should I denature the microRNA Marker before loading on my gel?
- What is the difference between the siRNA marker and the microRNA marker (NEB #N2101S)?
- Can I use EtBr for staining the microRNA Marker after gel electrophoresis?
- Should I load more of the microRNA Marker so I can get better visibility of the bands after staining the gel with EtBr?