What’s the DNA and RNA 3’- extension protocol and how to detect it with Urea-PAGE (Sequencing gel) (Ref 1)?

There are two distinct 3’-extension of RNA experiments. The first is known as “promoter independent” RNA extension. The second is known as “promoter-driven” RNA extension.

Materials and Instruments

• Instruments

• DNA oligonucleotides (Provided):

>Non-template-oligo

5-AATTAATACGACTCACTATAGGAATAAGTAGAGGTGAAGATTTA-3

>Template-oligo

3-TTAATTATGCTGAGTGATATCCTTATTCATCTCCACTTCTAAAT-5

• Synthetic 24mer RNA Solution (20μM):

>s24mer-RNA

5-GGAAUAAGUAGAGGUGAAGAUUUA-3

1 OD powder of Synthetic RNA is provided. Add 184.5 μL water in the tube.

• Dimethyl staining solution (this can be reusable):

0.4M NaOAc, 0.2g/100mL dimethyl blue (power provided), pH=4.7

• 10x TBE buffer (Not Provided):

108g Tris-HCI, 7.44g EDTA, 55g Boric Acid, pH=8.3, dissolved in 1L water

• 15% Urea-PAGE (Not Provided) (For 20mL):

How to make 15% Urea-PAGE Gel?

1. Place the two pieces of glass together and seal the edges and bottom to create a gel mold.
2. Prepare the 15% Urea-PAGE and add APS and TEMED, according to the manufacturer's instructions [National diagnostics].
3. Quickly transfer the Urea-PAGE into the gel mold and place a comb on top to create wells for sample loading.
4. Place the gel in the running box and fill it with 1x TBE buffer.
5. Pre-run the gel at a constant 12W for 30 minutes to ensure even separation.
6. Using a syringe, carefully wash out any remaining urea in the wells.
7. The Urea-PAGE is now ready to be used for sample separation.

Experimental Procedure

RNA 3’ extension (IVT)

1. Prepare RNA 3' extension IVT reaction mix accordingly.
2. Perform RNA 3' extension IVT in an IVT system at 37°C for 2 hours.
3. After incubation, add 2-5μL of the IVT product to a pre-run 15% urea-PAGE.
4. Run the gel at a constant voltage of 12W for 1-2 hours.
5. Stain the gel with dimethyl staining solution for 5 minutes, and then de-stain it with ddH2O until the dye disappears.
6. Visualize the gel in the Coomassie blue channel on a Gel Imaging System (Bio-Rad, Chemidoc).

DNA 3’ extension (IVT)

1. Prepare an oligo DNA template by annealing two oligo nucleotides.
2. Prepare RNA 3' extension IVT reaction mix accordingly.
3. Perform RNA 3' extension IVT in an IVT system at 37°C for 2-3 hours.
4. DNase I (4U/μl) digestion to remove DNA template at 37°C for 30 minutes. 5. After incubation, add 2-5μL of the IVT product to a pre-run 15% urea-PAGE.
5. Run the gel at a constant voltage of 12W for 1-2 hours.
6. Stain the gel with dimethyl staining solution for 5 minutes, and then de-stain it with ddH2O until the dye disappears.
7. Visualize the gel in the Coomassie blue channel on a Gel Imaging System (Bio-Rad, Chemidoc).

1. Gholamalipour, , Karunanayake Mudiyanselage, A. & Martin, C.T. 3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character- RNA-Seq analyses. Nucleic Acids Res 46, 9253-9263 (2018).