Preparation of dsRNA by In Vitro Transcription Protocol

Experiment Summary

This protocol is a simple and widely used in vitro transcription reaction for the preparation of double-stranded RNA using PCR templates. The dsRNA prepared by this method can be used to induce RNA interference in certain cells or tissues.

Materials and Reagents

Agarose gel (1 %)
Recovery buffer (10 X)
ATP, CTP, GTP, UTP (100 mmol/L each)
Dithiothreitol (DTT) (1mol/L)
DNA molecular quality standards
dNTP mixture, 10 mmol/L each
Ethanol (100% and 70%)
Ficoll-Orange G Sampling Buffer (6 X)
Non-Denaturing Gel Sampling Buffer (10 X)
Nuclease-free water
Oligonucleic acid primers
PCR Buffer (10 X)
Phenol: chloroform (1: 1, VIV)
Pancreatic DNAase I without RNAase (2U/μL)
Sodium acetate (3mol/L, pH 5.2)
SYBR Gold (10000 X) or ethidium bromide (10mg/mL)
T7RNA polymerase (20U/μL)
T7 Transcription Buffer (10 X)
TAE Buffer (50 X, 1 X)
Taq DNA Polymerase (2.5U/μL)
TBE buffer (5 X, 0.5 X)
DNA template for PCR

Equipment

Agarose gel electrophoresis instrument
Centrifuge
Microcentrifuge tube (1.5m.L)
PCR tubes (0.5mL, thin-walled)
Primer3 software
Spectrophotometer
PCR instrument
Vortex oscillator

Procedure

A. Preparation of DNA templates for in vitro transcription

Analyze the mRNA, cDNA or genomic sequence of the target gene in the relevant database or according to the sequence data.
Select the target region with a length of 500-800 bp as the dsRNA template.
Use BLASTn to analyze the homology of the sense and antisense strands. If a strand is homologous to a non-target gene, repeat step 2.
Forward and reverse primers of 20 ~ 24 nucleic acids in length were designed using Primer3 software to have a Tm value of approximately 60°C.
The T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') was added to the 5' end of both primers. It is necessary to prepare forward and reverse primers (two pairs of primers) with or without the T7 promoter sequence.

Two separate PCR reactions are performed to generate DNA templates for the RNA sense and antisense strands. For each DNA template generated, the following reagents are required to make up a 500 μL PCR system in a 1.5 mL tube:

Template DNA	-a
Nuclease free water Add to	395 μL
PCR buffer (10 X)	50μL
dNTP mix (10 mmol each)	10 μL
Forward Primer (10μmol)	20μL
Reverse primer (10μmol )	20μL
Taq DNA polymerase	5μL (12.5U)
Total reaction volume	500μL

a as a template for PCR, use 0.05~100ng of cloned plasmid or phage DNA, 0.5~5 μg of genomic DNA, or 10~20 μL of cDNA generated by the reverse transcription reaction.

Mix lightly and centrifuge for 1-2 s. Shake the reagents to the bottom of the tubes and dispense 100 μL into 0.5 mL thin-walled PCR tubes.
Place the tubes in a thermal cycler and perform 25 amplification cycles according to the following program:
i. 94 °C 2min
ii. 94°C 45s
iii. 50°C 45s
iv. 72°C 60s
v. Repeat ii~iv again, 24 cycles.
vi. 72 °C 5min
vii. Store at 4 °C
viii. End.
At the same time, a 1% agarose gel was prepared with TAE buffer.
At the end of the PCR reaction, 5 μL of PCR product was mixed with 1 μL of 6X Ficoll Orange G Sampling Buffer and the product was analyzed by electrophoresis; the Orange G dye was located at the front of the electrophoresis and did not obscure any of the bands during electrophoresis.
Transfer the remaining DNA amplification product from step 10 to a new 1.5 mL microcentrifuge tube, add 1/10 volume of sodium acetate (3 mol/L, pH 5.2) and 2.5 times the volume of anhydrous ethanol, vortex well, and allow to settle the PCR product for more than 30 min at -20°C or lower. Centrifuge the product for 30 min at 4°C maximum speed. Centrifuge for 30 min at maximal speed at 4°C and discard the supernatant.
Wash the precipitate with 1mL of 70% ethanol to remove residual salts. centrifuge at 4 °C for 5min at maximum speed, discard as much of the supernatant (70% ethanol) as possible, and leave the tube uncapped for a few minutes to allow the ethanol to evaporate.
Dissolve the DNA precipitate in 50 μL of water.

B. Preparation of dsRNA

Place T7RNA polymerase on ice and other reagents at room temperature. At room temperature, prepare a 100 μL in vitro transcription system in a 1.5 mL microcentrifuge tube according to the following recipe:

Nuclease-free water	56.5 μL
T7 transcription buffer (10 X)	10μL
PCR template DNA	5μL
ATP (100mmol/L)	5μL
CTP (100mmol/L)	5μL
UTP (100mmol/L)	5μL
GTP (100mmol/L)	8μL
OTT (1mol/L)	0.5μL
T7RNA polymerase	5μL (100U)

If the experiment requires more RNA, expand the reaction system proportionally.

Gently vortex for 1 ~ 2s to centrifuge the reagent to the bottom of the tube.
Incubate at 37°C for 2h.
Add 5 μL (10 U) of RNase-free pancreatic DNase I. Incubate at 37°C for 2 h. Add 5 μL (10 U) of RNAase-free pancreatic DNase I.
Gently vortex for 1~2s to dump the reagent to the bottom of the tube.
Incubate at 37°C for 30 min.
Add an equal volume of phenol:chloroform (1: 1, VIV) and vortex for 20 s. Centrifuge at 4°C for 15 min at maximum speed to separate the solid phase from the liquid phase, and transfer the liquid phase to a new 1.5 mL microcentrifuge tube.
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 to stand at -20°C or lower for 30 min or more to allow the PCR product to precipitate. centrifuge the product at 4°C for 30 min at maximum speed.
Discard the supernatant. Add 1mL of 70% ethanol to wash the precipitate to remove residual salts. centrifuge for 5min at 4 °C max to precipitate the RNA, discard the supernatant (70% ethanol) to the maximum extent possible, and leave the tube uncapped for several minutes to allow the ethanol to evaporate.
The RNA precipitate was dissolved in 100 μL of nuclease-free water. The concentration of RNA was measured using an absorption light spectrophotometer.
The two strands were denatured to produce a 0.5 μmol/L dsRNA solution:
Positive RNA 50pmol
Antisense RNA 50pmol
Replication buffer (10 X) 10 μL
Nuclease-free water to 100 μL
Place the tube in a thermal cycler at 95°C for 1min, turn off the heat source and allow the temperature to slowly decrease to room temperature.
Precipitate RNA as in steps 8 and 9.
Dissolve the precipitate in water to make a stock solution of lμg/μL and store at -80 °C.

C. Checking the integrity of dsRNA

Prepare a 1 % agarose gel with 0.5 X TBE buffer.
Dissolve dsRNA (obtained in step B.14) and ssRNA (obtained in step B.10 for both the sense and antisense strands, used as a control) in 1 X nondenaturing gel-on-sample buffer at a final concentration of 0.1 μg/μL.
5 μL of each sample was injected into 0.5 X TBE buffer and electrophoresed at a constant voltage of 75 V. After electrophoresis, the samples were separated from each other at room temperature.
After electrophoresis, the samples were stained with SYBR Gold (1: 10,000 in 0.5 X TBE buffer) or ethidium bromide (1: 20,000 in 0.5 X TBE buffer) for 10 min at room temperature, and the RNA bands were visualized under ultraviolet light.

* For research use only. Not intended for any clinical use.

Quick Inquiry

Positive RNA	50pmol
Antisense RNA	50pmol
Replication buffer (10 X)	10 μL
Nuclease-free water	to 100 μL

Preparation of dsRNA by In Vitro Transcription Protocol

Experiment Summary

Materials and Reagents

Equipment

Procedure

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