RNA Interference All small interference RNAs (siRNAs) and scrambled control (Sb) are from Ambion except an additional scramble control corresponding to the siControl Non-targeting siRNA Pool
(D001206-13-05) from Dharmacon. siRNAs: Sb:Silencer Negative Control #1 siRNA (AM4611), #2 siRNA (AM4613) and #3 (AM4615). si?Actin:Silencer ?-actin siRNA (AM4607), siGAPDH:Silencer GAPDH siRNA (AM4624). The siRNAs sequence (5’-3’) and targeting exons are shown:
Overview of siRNA Transfection Protocol
Design the siRNA concentration Stock 20 uM Final 30 nM with medium 2 ml/6well-plate+96w (4xwell) C1V1=C2V2 10 nM : 20 uM x ? ul = 10 nM x 2 ml ? = 1ul 30 nM : 20 uM x ? ul = 30 nM x 2 ml ? = 3ul Step 0: Warm up medium/trypsin etc and take siRNA etc out Label 6x 0.5 ml epp, and 1.5 ml epp. Add the amount of OPT medium, then Add proper Neo(make master mix) or siRNA to each epp.
Step 1 Detach cells＋ preparevehicle master mix (A) and siRNA mix (B), then incubate 5-10 min RT
A. transfection vehicle
Master mix =75ul OPT × 5＋ 4ul Neo × 5
B. siRNA mix
110nM 2Neo 10nM 3Sb 4Sb 5N2 6N2
75ul OPT +siRNA
Step 2 prepare siRNA vehicle complex by mix 75ul A to B(75ul)=150ul , then incubate 10 min RT
75ul Complex 150ul 75ul
12Neo 3Sb 4Sb 5N2 6N2
? ? ?
Count cell, and prepare cell to make final concentration 0.1 Million/ml suspension in medium Add 150 ul siRNA complex to plate, then add 1ml (= 0.1 M cell suspension), Top up with 0.85 ml medium. Total: 150ul +1 ml + 0.85 ml= 2 ml
Sub-confluent cells were detached and transfected with siRNAs using the siPORT? NeoFX? Transfection Agent (AM4511, Ambion). For HaCaT cells, siRNA was diluted in 75 ?l OPT medium and NeoFX 3 ?l in 75 ?l OPT medium respectively and was incubated 10 mins at RT, then mixed well, and incubated at RT for a further 10 mins to allow the formation of siRNA complexes. The siRNA complex was then placed in 6-well plates, and 0.6~1 x 105 cells in 1.5 ml of the normal cell growth medium were added to give a total volume of 1.65 ml. For FEK4 cells, 200 ?l of the siRNA complex: 4 ?l NeoFX in 100 ?l of OPT medium + siRNA in 100 ?l OPT medium) were added to a 60-mm plate, with 2 x 105 cells in 2 ml of the normal cell grown medium to give a total volume of 2.2 ml. The following day, 0.5 volumes of fresh medium was added and cells were incubated untill further treatment.
Can change medium ~12h after seeding if cytotoxicity observed. Or no antibiotics. Need assay knock down and cell viability. 48h for mRNA and 72h for protein. Cell viability can be check by GAPDH Assay or cell necrosis and apoptosis. > 70% knock down and <15% cell death. Or Trypan blue exclusion assay. So 48-72h (better) after seeding/transfection, coverslips were collected and left cells were detached by trypsin and resuspend in 10%FCS medium-PBS, take a portion for cell count (Trypan blue) and left spin down and wash then lysis with 50ul NP-40 lysis buffer (plus sample buffer? Since there may not enough cells)collect cells for protein lysate.
siHO2: NM_002134. # 1: ID 11243, Exon 3; #2: ID 117046, Exon 3,4.
sense #1 HO2 #2 HO2 antisense sense antisense
GGACAUGGAGUAUUUCUUUtt AAAGAAAUACUCCAUGUCCtt CCAAAGAGAGGAUCGUGGAtt UCCACGAUCCUCUCUUUGGtc
siHO1: NM_002133. #1: ID 11242, Exon 5; #2: ID 11056 Exon 2.
sense #1 HO1 #2 HO1 antisense sense antisense GGCCUUCUUUCUAGAGAGGtt CCUCUCUAGAAAGAAGGCCtt GGCAGAGAAUGCUGAGUUCtt GAACUCAGCAUUCUCUGCCtg
siNrf2: NM_006164. #1: ID 115764, Exon 5; #2: ID 115763, Exon 3.
sense #1 Nrf2 #2 Nrf2 antisense sense antisense CCUUAUAUCUCGAAGUUUUtt AAAACUUCGAGAUAUAAGGtg GCUUUUGGCGCAGACAUUCtt GAAUGUCUGCGCCAAAAGCtg
siBach 1: NM_001011545. #1: ID 115188, Exon 3, #2: ID 115189, Exon 5.
sense #1 Bach1 #2 Bach1 antisense sense antisense GCCUUUGUCAGGUACAGACtt GUCUGUACCUGACAAAGGCtt CCAUCUAAUUUUCUCCUGAtt UCAGGAGAAAAUUAGAUGGtt
The Mechanism of RNA Interference (RNAi)
Long double-stranded RNAs (dsRNAs; typically >200 nt) can be used to silence the expression of target genes. Upon introduction, the long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway. First, the dsRNAs get processed into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer (initiation step). Then, the siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand.
RNA Interference and RNA Silencing
RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequencespecific manner. In all RNA silencing pathways, dsRNA is processed to a 21–30 nucleotide-long RNA, which then functions as a component of a “silencing complex” to specifically repress expression or function of a target gene or genomic region Specifically, in the RNAi pathway, dsRNA is processed to short interfering RNA (siRNA): 21–25 bp dsRNA with dinucleotide 3' overhangs. One strand of the siRNA (the guide strand) is then assembled into an RNA-induced silencing complex (RISC) that cleaves the target mRNA. The siRNA is thought to provide target specificity to RISC through base pairing of the guide strand with the target mRNA
In vitro generation of siRNA
In vitro Transcription (T7 RNA polymerase Long RNA
PCR primers including T7 minimal promoter
Anneal Long dsRNA
RNase III treatment siRNA – 18-25bp