In this protocol the design and production of siRNA via In Vitro Transcription (IVT) is described.
Small interfering RNA (siRNA) are 21–23 nt dsRNA to be used for knock-down of gene expression through RNAi.
| Item | Item article number | Distributor | Vendor |
|---|---|---|---|
| HiScribe T7 High Yield RNA Synthesis Kit |
E2040S | NEB | NEB |
| UltraPure™ DNase/RNase-Free Distilled Water | 10977035 | Thermofisher | Invitrogen |
| Klenow exo- (5U/μl) | EP0421 | ThermoFisher | ThermoFisher |
| RNase T | EN0541 | ThermoFisher | ThermoFisher |
| DNase I, RNase-free (1 U/µL) | EN0521 |
ThermoFisher | ThermoFisher |
| HiScribe T7 High Yield RNA Synthesis Kit | E2040S | NEB | NEB |
| optional: 5-Ethynyl-UTP (for fluorescent labeling) | CLK-T08-S | Jena Biosciences | Jena Biosciences |
Find 21 nt sequences in the target mRNA that begin with an AA dinucleotide.
Have all oligos at 100 μM stock concentration in nuclease-free water.
1️⃣ Make the following two mixtures:
| Reagent | Volume | Final concentration |
|---|---|---|
| Nuclease-free water | 5 μl | |
| 10x Klenow buffer | 1 μl | 1x |
| T7 RNA promotor handle (100 μM) | 2 μl | 20 μM |
| Sense template (100 μM) | 2 μl | 20 μM |
| TOTAL: | 10 μl |
and for antisense template:
| Reagent | Volume | Final concentration |
|---|---|---|
| Nuclease-free water | 5 μl | |
| 10x Klenow buffer | 1 μl | 1x |
| T7 RNA promotor handle (100 μM) | 2 μl | 20 μM |
| Antisense template (100 μM) | 2 μl | 20 μM |
| TOTAL: | 10 μl |
2️⃣ Heat for 5 min at 70°C 🔥and leave to anneal at room temperature for a minimum of 5 mins.
3️⃣ Supplement the 10 μl template mixtures with a Klenow reaction.
| Reagent | Volume | Final concentration |
|---|---|---|
| Hybridization mixture from previous step | 10 μl | |
| Nuclease-free water | 6.5 μl | |
| 10x Klenow buffer | 1 μl | 1x |
| dNTP (10 mM) | 0.5 μl | 250 μM |
| Klenow polymerase, exo-, (5 U/µL) | 2 μl | 0.5 U/µL |
| TOTAL: | 20 μl |
4️⃣ Incubate at 37°C for 30 min ⏰.
PAUSE POINT: Proceed to dsRNA synthesis, or store the templates at –20°C
5️⃣ Assemble the transcription mixture (optional to include 5-Ethynyl-UTP, can be used for fluorescently label the RNA to confirm transfection by microscopy). Make one transcription reaction for each template (sense vs. antisense):
| Reagent | Volume | Final concentration |
|---|---|---|
| Nuclease-free water | 7 µl | |
| 10X reaction buffer (B2041) | 1.5 µl | 0.75X |
| ATP (100 mM) | 1.5 µl | 7.5 mM |
| GTP (100 mM) | 1.5 µl | 7.5 mM |
| CTP (100 mM) | 1.5 µl | 7.5 mM |
| UTP (100 mM) | 1 µl | 5 mM |
| 5-Ethynyl-UTP (❗️10 mM) | 2.5 µl | 1.25 mM |
| Template (0.5 µg/µl), from previous step | 2 µl | 1 µg dsDNA |
| T7 RNA Polymerase Mix (M0255) | 1.5 µl | |
| TOTAL: | 20 µl |
6️⃣ Incubate reactions 2 hr at 37°C.
7️⃣ Combine the sense and antisense transcription reactions and incubate at 37°C overnight.
8️⃣ Add RNase T and DNase and incubate for 2 hr at 37°C:
| Reagent | Volume | Final concentration |
|---|---|---|
| IVT reaction from previous step | 40 μl | |
| Nuclease-free water | 44.5 μl | |
| 10x RNAse T buffer | 10 μl | 1x |
| RNase T | 3 μl | |
| DNase | 2.5 μl | |
| TOTAL: | 100 μl |
9️⃣ Purify the product through either RNA column or oligonucleotide column.
Elute to 51 μl.
🔟 Quantification. The siRNA concentration used for transfection is critical to the success of gene silencing experiments.
Transfecting too much siRNA causes nonspecific reductions in gene expression and toxicity to the transfected cells.
Transfecting too little siRNA does not change the expression of the target gene.
Measure the A260 on NanoDrop of a 1:25 dilution of the siRNA product.
Enter the A260 reading in the following formula to get concentration: