Preparation of dsRNA
Principle
Two complementary RNA strands are transcribed in vitro from the DNA template of the target gene, and the resulting RNA strand is subsequently annealed to form dsRNA.
Operation method
Synthesis of dsRNA by in vitro transcription
Principle
Two complementary RNA strands are transcribed in vitro from the DNA template of the target gene, and the resulting RNA strand is subsequently annealed to form dsRNA.
Materials and Instruments
Reagents: Move I. Preparation of templates 1、Consult the gene sequence library and download the sequence of the target gene. 2. Select the target gene sequence of 400-800 bp as the template for in vitro transcription. 3. Conduct BLAST comparison to analyze the specificity of the sequences, if the specificity is not good, we need to re-select the sequences. 4. Use Primer5.0 to design primers for the target gene. 5. Add the T7 promoter sequence (5′-TAATACGACTCACTATAGGG-3′) to the 5' end of the two primers, and prepare two pairs of primers with or without the T7 promoter sequence. 6. PCR was performed with each primer pair to synthesize DNA templates, and the PCR system was configured in 200 μl of RNAse free EP tubes: Template DNA Genomic DNA: 6 ug RNase Free dH2O Add to 100 μl SYBR Taq enzyme (2×) 50 μl Forward primer (10 μM) 5 μl Reverse primer (10 μM) 5 μl Total reaction volume 100 μl 7. After mixing, vortex and centrifuge to the bottom of the tube, put it into the PCR instrument and carry out amplification as follows: Cycle Cycle Point Hold 94 ℃, 5 min Cycling (25 repeats) Step1: 94 ℃, hold 30 s Step2：64 ℃, hold 30 s Step3：72 ℃, hold 10 s Hold 72 ℃, 5 min Hold 72 ℃, 5 min 8、Preparation of 1% agarose gel electrophoresis
Agarose gel, electrophoresis buffer, ATP, CTP, GTP, UTP, DTT, DNA marker, OTT, High Fidelity PCR Mix, dNTP mix, anhydrous ethanol, DNA uploading buffer (6X), TBE (5X), RNase free water, primers, PCR buffer, PCR tubes, enzyme free EP tubes of various sizes, Phenol : Chloroform (1 : 1), DNAase I, Sodium Acetate, Gold View Nucleic Acid Stain, T7 Polymerase, T7 Transcription Buffer (10X), Whole Genome DNA.
Equipment:
Nucleic acid electrophoresis, PCR instrument, metal bath, gel imaging system.
9. At the end of the reaction, 5 μl of the PCR product was mixed with 1 μl of DNA buffer (6X) and added to the sample wells for electrophoresis.
10. The remaining PCR product was transferred to a new 800-μl EP tube and vortexed with 1/10 of a volume of sodium acetate (3 mol/L, pH 5.2) and 2.5 times the volume of anhydrous ethanol and allowed to settle for 30 min or more at -20 °C or lower. The supernatant was centrifuged at 16,000g for 30 min at 4 °C. The product was washed with 1 ml of 70% ethanol and the supernatant was discarded. Wash the precipitate with 1 ml of 70% ethanol to remove residual salts, centrifuge at 16,000g for 5 min at 4 °C, remove as much supernatant as possible, and leave the tube uncapped for several minutes to allow the ethanol to evaporate.
11. Dissolve the DNA precipitate in 50 μl of water.
Preparation of dsRNA
1. Place T7RNA polymerase on ice and configure 100 μl of in vitro transcription system according to the table below:
RNase Free dH2O | 56.5 μl |
T7 Transcription Buffer (10X) | 10 μl |
PCR template DNA | 5 μl |
ATP (100 mmol/L) | 5 μl |
CTP (100 mmol/L) | 5 μl |
UTP (100 mmol/L) | 5 μl |
GTP (100 mmol/L) | 8 μl |
OTT (1 mol/L) | 0.5 μl |
T7RNA polymerase | 0.5 μl (100 U) |
2. Centrifuge the reagents at the bottom of the EP tube and incubate at 37 ℃ for 120 min.
3. Add 5 μl of DNAase I, vortex it to the bottom of the tube and incubate at 37 ℃ for 30 min.
4. Add an equal volume of phenol:chloroform (1:1), vortex for 30 s. Centrifuge at maximum speed for 15 min at 4 ℃ and transfer the upper liquid phase to a new 1.5 ml microcentrifuge tube.
5. Add 1/10 volume of sodium acetate (3 mol/L, pH 5.2) and 2.5 times the volume of anhydrous ethanol to the liquid phase, vortex well, and allow the PCR product to precipitate for more than 30 min at -20 ℃. centrifuge the supernatant at 16,000 ℃ for 30 min.
6. Discard the supernatant. Wash the precipitate with 1 ml of 70% ethanol to remove residual salts, centrifuge at 16,000 °C for 5 min at 4 °C to precipitate the RNA, discard as much of the supernatant as possible (70% ethanol), and leave the tube uncapped for several minutes to allow the ethanol to evaporate.
7. Dissolve the RNA precipitate in 100 μl of nuclease-free water. Measure the concentration and purity of the RNA using a Nanodrop.
8. The two strands are denatured to produce a 0.5 μmol/L solution of dsRNA:
9. Place the EP tube in a metal bath at 95°C for 1 min and cool naturally to room temperature.
10. Add 1/10 volume of sodium acetate (3 mol/L, pH 5.2) and 2.5 times the volume of anhydrous ethanol to the liquid phase, vortex to mix well, and allow the PCR product to precipitate for more than 30 min at -20 ℃. centrifuge the product at 16,000 g for 30 min at 4 ℃. discard the supernatant. The supernatant is discarded and the precipitate is washed with 1 ml of 70% ethanol to remove residual salts. The RNA is precipitated by centrifugation at 16,000g for 5 min at 4 °C, the supernatant is discarded to the maximum extent possible (70% ethanol), and the centrifuge tubes are uncapped and left for a few minutes to allow the ethanol to evaporate.
11. Dissolve the RNA with RNase free water and store at -80 ℃.
dsRNA integrity test
1、Prepare 1% agarose gel electrophoresis.
2. After the reaction, mix 5 μl of PCR product with 1 μl of DNA buffer (6X) and add to the sample wells.
3. The gel was removed and color developed in Gold View Nucleic Acid Stain, then rinsed three times in 1X TBE for 5 min each time and assayed on the machine.
Caveat
1、The concentration of RNA is too low when extracting RNA, you can prolong the precipitation time appropriately during the extraction process, precipitate overnight, or add a trace amount of glycogen.
2. If other bands are found in the process of dsRNA integrity testing, it may be that the RNA degraded or transcribed RNA is not specific.
3， the target gene sequence should not be too long, otherwise the specificity will be reduced, and the contamination of stray bands will occur easily. Common Problems Question 1: Is the concentration too low during RNA extraction? A: You can appropriately increase the template concentration, extend the in vitro transcription time, as well as appropriately lengthen the precipitation time during the extraction process and precipitate overnight. Q2: What are the stray bands found during dsRNA integrity testing? A: RNAase-free reagents, EP tubes and pipette tips are used to avoid degradation of the target dsRNA. For more product details, please visit Aladdin Scientific website.