RNA interference technology

RNA interference (RNAi) is a cellular mechanism that suppresses gene expression by inhibiting gene transcription or activating RNA degradation processes. This mechanism was discovered in plants in 1998 by Andrew Fire and Craig Mello. Today, this phenomenon is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technology, providing an effective method to identify and determine the function of multiple genes without genetically modifying the organism. RNAi is instrumental in analyzing gene function. For example, RNAi technology helped screen chromosomes I and III of nematodes and identify genes involved in cell division and embryonic development. The technology has also been successfully applied to fruit flies to identify genes that play crucial roles in embryonic development, biochemical signaling cascades, and other fundamental cellular processes. In coffee plants, RNA constructs were used to knock down the gene responsible for producing theobromine synthase, resulting in decaffeinated coffee plants. Studies have shown that small interfering RNAs (siRNAs) can suppress infections caused by human immunodeficiency virus, hepatitis B virus, and poliovirus in human cell lines. Researchers have also successfully knocked down genes expressing respiratory syncytial virus, which causes severe respiratory illness in infants and newborns. Before the discovery of RNAi technology, gene function was analyzed by knocking out the gene of interest from the genome and observing phenotypic changes. Gene knockout is an irreversible method, while RNAi is a reversible method that can silence protein-coding genes in the genome on a large scale. Furthermore, it is a precise technology that can differentially silence genes even with single-nucleotide variations. Therefore, it can help target dominant mutants, such as some oncogenes. In addition, compared to oligonucleotides or ribozymes used in older methods, RNAi technology is highly efficient because the effector molecules function at lower concentrations.

Constructing RNAi plasmids using pFGC5941
Amplifying cDNA or gDNA inserts using Phusion PCR

1. Amplify the cDNA or gDNA insert (if the fragment does not contain introns) using Phusion PCR.
Perform the following two 20 µL reactions in separate tubes:
Total volume (20 µL): 5x Phusion buffer (4 µL) + 10 mM dNTP (0.5 µL) + DMSO (0.6 µL) + Template (1.0 µL) + Phusion enzyme (0.2 µL) + dH2O (11.0 µL) + 5 µM forward primer (1.5 µL) + 5 µM reverse primer (1.5 µL)
2. Run the Phusion PCR program:
Digestion 3. Digest one insert fragment with NcoI/AscI and the other insert fragment with BamHI/XbaI.
Total volume (25 µL): 10x CutSmart buffer (2.5 µL) + dH2O (4.5 µL) + NcoI (1.5 µL) + AscI (1.5 µL) + PCR product (15 µL)
3.1 Incubate the sample at 37°C for 1 hour.
3.2 Gel purify the digest and save the BamHI/XbaI digested insert fragment for the second ligation.
First Ligation
4. First ligation (aim for a molar ratio of insert to vector of 2:1~6:1)
Total volume (20 µL): Linearized pFGC5941 digested with AscI/NcoI (~175ng; adjust volume as needed) (2 µL) + Insert fragment (digested with AscI/NcoI) (~15-30ng) (4 µL) + T4 ligase buffer (2 µL) + T4 ligase (1 µL) + dH2O (11 µL)
4.1 Incubate at room temperature for 30 minutes.
4.2 Transfer to... 10 µL transformed into competent E. coli cells (self-made) and plated on Kan plates.
Colony PCR check of the first insert
5. Colony PCR check of the first insert Total (10 µL): dH2O (8.0 µL) + 10x buffer (1.0 µL) + dNTP (0.125 µL) + pFGC5941 2372 F (0.5 µL) + pFGC5941 3082 R (0.5 µL) + Taq (0.05 µL)
5.1 Run colony PCR
6. Circle the largest colonies on the plate and label them 1-8
7. Make a replica plate of your colonies
8. Perform PCR on the first insert using primers on the vector to check the insert:
Empty vector will give a 700bp band
Colony selection and plasmid preparation
9. Pick two correct colonies and inoculate into 3 mL LB+Kan broth
10. Incubate overnight at 37℃ with shaking
11. The next day, perform plasmid preparation (mini-prep kit) using one well-grown colony
Digest plasmid with BamHI/XbaI
12. Total volume (50 µL): 10x CutSmart buffer (5 µL) + dH₂O (12 µL) + XbaI (1.5 µL) + BamHI + (1.5 µL) + plasmid (30 µL)
* Adjust volume according to concentration; you need 2000-5000 ng of plasmid
12.1 Incubate at 37℃ for 1 hour
12.2 Gel Purification and Digestion
Ligation #2
13. 2 µL of vector containing the first insert, digested with BamHI/XbaI (~175 ng; adjust volume based on concentration)
Total (20 µL): Insert digested with BamHI/XbaI (done in step 3) (~15-30 ng required) (4 µL) + T4 ligase buffer (2 µL) + T4 ligase (1 µL) + dH₂O (11 µL)
13.1 Incubate at room temperature for 30 minutes.
13.2 Transform 10 µL into competent E. coli cells (homemade) and plate on Kan plates.
Colony PCR to check for the second insert
14. pFGC5941 3930 F and pFGC5941 4430 R
Vector without insert will give a 500bp band.
15. Pick two correct colonies and inoculate into 3mL LB+Kan broth.
15.1 Incubate overnight at 37℃ with shaking.
15.2 Plasmid preparation (mini-prep kit)
Check plasmid for insert
16. PCR check for both inserts:
2372F/3082R or RNAi_R (insert specific)
3930F/4430R or RNAi_F (insert specific)
Sequence Verification
17. Use 4 primers:
2372F, 3082R, 3930F, 4430R
Note: Add DMSO to the sequencing reaction. To aid in sequencing across restrictive endonuclease digestion sites (chromatographic peaks typically drop sharply after the digestion site; another strategy is to use 2372F&3082R to PCR the left insert fragment of the final plasmid, and 3930F&4430R for the correct insert, and then sequence the PCR products). 18. Transformation into Agrobacterium for infiltration is sufficient.