Preparation of antisense RNA
RNA can also be synthesized in vitro by RNA polymerases (e.g., T3, T7, or SP6 ) using linear DNA as a template. This experiment is based on the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Antisense RNA preparation experiments
Materials and Instruments
Restriction Endonuclease Enzymatic Hydrolysis Template DNA E. coli DNA Polymerase Pancreatic DNase I Move I. Chemically synthesized RNA For more product details, please visit Aladdin Scientific website.
Chloroform Phenol Ethanol DTTrNTP solution Transcription buffer Tris-Cl Spermidine hydrochloride NaCl Placental RNase inhibitor Bovine serum albumin RNA polymerase DEPC
DNA synthesizer Agarose gel electrophoresis Microcentrifuge tube
RNA can also be used to detect functional structural domains of target genes or to regulate gene expression. Antisense RNA is complementary to specific structural domains of the target gene and can effectively form stable target RNA-antisense RNA hybrids.
1. RNA can be synthesized by automated DNA synthesizers, such as Applied Biosystem's DNA Synthesizer.
2. Cytosine is usually paired with 2'-OMe ( methoxy) RNA and 2'-OMe hypoxanthine instead of 2'-OMe guanine.
Preparation of Antisense RNA from Vector/cDNA Cloning and Transcription
RNA can also be synthesized in vitro by RNA polymerases (e.g., T3, T7, or SP6 ) using linear DNA as a template.
1. A DNA sequence is inserted into the polyclonal site of the vector, which carries a phage RNA polymerase transcriptional recognition sequence near the polyclonal site, i.e., with a T3, T7, or SP6 promoter (the DNA sequence cloned in the vector is the same as the target gene, and the transcription product is produced by transcribing from the 3' end of the promoter in the vector, so that the transcriptional product RNA is complementary to the target gene). Complementarity.
2. Prepare 2 pmol/L of template DNA (1 pmol/L of 3 kb plasmid is about 2 μg) by enzymatic hydrolysis with appropriate restriction endonuclease, and take a portion of the hydrolyzed DNA (about 100 ng is needed) for agarose gel electrophoresis analysis.
3. The protruding end of the 3' end of the DNA can be removed by Klenow fragment of E. coli DNA polymerase or phage T4 DNA polymerase in the presence of 4 dNTP.
4. The template DNA is purified with chloroform:phenol and then precipitated with ethanol. The DNA is then dissolved in TE (pH 7.0) at a concentration of approximately 250-500 nmol/L. The DNA is then extracted by chloroform.
5. At room temperature, mix the following components sequentially in a microcentrifuge tube: 0.2 pmol of DNA template, 0.1 μl of DTT (1 mol/L), 1.0 μl of rNTP solution (5 mmol/L of each), 1.0 μl of 10X Transcription Buffer, 400 mmol/L Tris-Cl (pH 7.5 at 37°C), 60 mmol/L MgCI2, 20 mmol/L Spermidine hydrochloride, 20 mmol/L Spermine hydrochloride, 1.0 μl of Spermidine hydrochloride, and 0.0 μl of Spermine hydrochloride. 20 mmol/L spermidine hydrochloride, 50 mmol/L NaCl, placental RNase inhibitor (10U) 0.5 μl, bovine serum albumin (2 mg/ml) 0.5 μl, phage DNA-dependent RNA polymerase (10U) 1.0 μl, and replenished to 10 μl with DEPC water, all of the above buffers should be sterilized and stored in separate tubes at -20°C.
The above buffers should be sterilized and stored in separate tubes at -20°C. Avoid air bubbles when mixing the components. Place the reaction solution at 37°C for 1~2 h (T3 or T7 RNA polymerase) or 40°C for 1~2 h (SP6 RNA polymerase).
6. Add 1 μl of tryptic DNase Ⅰ (1 U/μl) without RNase, mix well, and react at 37℃ for 15 min.
7. Add 100 μl of RNase-free water and purify RNA with 1:1 phenol:chloroform.
8. Transfer the aqueous phase to another centrifuge tube, add 20 μl of 5 mol/L ammonium acetate, mix well, and then add 250 μl of ice pre-cooled ethanol, let stand for 60 min at -20°C, centrifuge at 12000 g for 20 min at 4°C, and collect the RNA.
9. Discard the ethanol and allow the residual ethanol to evaporate. Dissolve the RNA precipitate in 100 μl of RNase-free water. Add 2 times the volume of ice pre-cooled ethanol, mix well and store in separate tubes at -70°C. Remove the tubes before use. Prior to use, remove the tube and add 0.1 volume of 5 mol/L ammonium acetate, mix well, place at -20°C for more than 15 minutes, centrifuge the precipitate at 12000 g at 4°C, remove the ethanol, and dissolve the RNA in an appropriate volume of the corresponding RNase-free buffer.