ABGENT CUSTOM SERVICES: RNAi Protocol Ⅰ. RNAi Introduction A. RNAi Workflow Solution B. RNAi Components Componets sirna Oligos Transfection Reagents Controls Monitering Gene Experssion Reagents Description Full Complement With mrna of Your Knock-down Target gene Such as Cation liposome, RNAi-Mate, Electricity Perforation Negetive Control, Positive control and Mocking transfecion mrna Expression In Transcriptional Level Monitoring of Target protein In Translation Level Ⅱ. sirna Design A. sirna Design In Efficiency of Gene Expression inhibition depends on selection target sequences. Target sequence may be selected at random, and also by specific sequence in different regions. Notice: sirna design in Mammalian, as follows: Page 1 of 6
1. Choose a region of 19 nucleotides. 2. Do not select sequences within the 5' and 3' untranslated regions (UTRs) or regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites (Elbashir et al., 2001). UTR-binding proteins and/or translation initiation complexes may interfere with binding of the RISC. 3. Do not select sequences that contain a consecutive run of 3 or more thymidine residues; a poly (T) tract within the sequence can potentially cause premature termination the shrna transcript. 4. Calculate the GC content of the selected 19-base oligonucleotide sequence. The GC content should be between 40% and 60%; a GC content of approximately 45% is ideal. 5. Sequences that have at least 3 A or T residues in positions 15 19 of the sense sequence also appear to have increased knockdown activity. 6. Check the 19-base oligonucleotide for secondary structure and long base runs, both of which can interfere with proper annealing. Eliminate candidate sequences that display these characteristics. 7. Compare the remaining candidate sequences to an appropriate genome database to identify sequences that are specific for the gene of interest and show no significant homology to other genes. Candidate sequences that meet these criteria are potential sirna target sites. 8. To optimize gene silencing, we highly recommend that you test more than one sirna target sequence per gene. 9. The results show that: UU 3 end and dtdt 3 end sirna have the same knock-donw efficiency. B. sirna Oligo Technical Data Notice: technical data related with sirna: 1. sirna MW 13,300. 2. Calculating sirna oligo OD, nmol and quality; Generally, for a 21 bp sirna oligo, have a simple relation,as follows:1 OD duplex=3.0 nmols=40 ug. 3. 1 OD sirna is diluted to 20 um, use 150 ul DEPC H 2 O to resuspend 1 OD sirna to make a 20 um solution. 4. Fluorescent Dye Labeled sirna Oligo RNA, such as FAM, HEX, and TAMRA should be protected from light. III. sirna Control A. Common Negative Control Page 2 of 6
We should choose sirna sequence which has the same composition as negative control sirna, but not obvious homology with mrna (scrambled control). The usual procedure is that we choose mismatch sirna sequence, which we must guarantees target do not have homology. 1. sirna experiment should have negative control; 2. Ready-to-use and haven t homologous sequence with target gene; 3. Scrambled sirna sequence which have the same composition as negative control sirna, but not obvious homology with mrna; 4. Negative control should haven t homologous sequence with other gene sequence. B. Fluorescent Dye Labeled sirna Negative Control Abgent 6-FAM negative control: Ready-to-use and haven t homologous sequence with target gene C. sirna Positive Control Positive control inspecting experimental system is very important. In other words, when you see sirna positive control anticipated experimental result, you can guarantee that yours experimental technique, transfection, RNA extraction and detection method are reliable. D. Mock Transfection 1. Abgent RNAi negative control does not have homology with mammal gene; 2. Conveniently observing transfection efficiency under fluorescence microscope; 3. NC-FAM advantageous to optimization of transfection condition; 4. The fluorescence is easy to photograph, it has very good ph tolerance, thus is stabler in the living cell Abgent Often used sirna positive control: 1 LaminA/C 2 GFP22 3 Luciferase GL2 4 MAPK1 5 Beta-Actin 6 Vimentin 7 P53 8 GAPDH 9 Cyclophilin B For one perfect control system, Mock Transfectionis necessary. Mock Transfectionmay examine Transfectionreagent influence on the toxicity of cell, survival rate and so on. We recommend using our company Abgent transfection reagent-rnai-mate. (See transfection reagent sheet) A good way to enhance confidence in RNAi data is to demonstrate a similar effect with two or more sirna targeted to different sites in the message under study. Page 3 of 6
E. Multiplicity Control Ⅳ. sirna Transfection A. sirna Transfection Methods At present, common method in mammalian transfection includes: Calcium phosphate co-precipitation, Electricity perforation law, DEAE-glycon and polybrene, machinery method (for example, microinjection and gene-gun), cation liposome reagent. Cation liposome reagent is most commonly used transfection method. B. RNAi-Mate Transfection Reagents Notice: Cation liposome transfection reagent should be: 1. Transfection reagents volumes 2. sirna Amounts 3. Cell density during transfection 4. Operation order during transfecion 5. The incubation time of cell and tranfection reagents/sirna complexes Transfection of primary culture and transformation of cell strain gene; High-throughput transfection of sirna /DNA tansfection; Co-transfection of DNA and sirna; In vivo delivery of sirna oligos(sirna,dna and RNA); Transfection of adherent cell and suspension cells. RNAi-Mate Applied field: Transfection of primary culture and transformation of cell strain gene High-throughput transfection of sirna DNA tansfection; Co-transfection of DNA and sirna In vivo delivery of sirna oligos (sirna,dna and RNA) Transfection of adherent cell and suspension cells RNAi-Mate Features: No necessary to change culture media. Easy to operate. Good repeatability. Transfect sirna oligos in high efficiency High trnasfection efficiency can be obtained even in culture media containing serum Ready to use,transfection in culture medium with antibiotics Cation liposome reagen ensure that No RNAase High transfection efficiency in vivo C. RNAi-Mate Applied Cell Lines RNAi-mate can be used for the transfection of DNA and sirna into many different kind cells. Our new sirna transfection reagents have been extensively tested in many different Page 4 of 6
kinds of cell lines originated from different sources, ranging from standard lines, e.g. HeLa, MC-7, Hep3B, COS-7, Neuro-2a, NIKS, B16, DLD-1, NIH/3T3, HT-29, A549, CHO-K1 and 293, and SVRbag4. D. Adherent Cell Transfection Procedure This procedure is suited to adherent cell transfection using 24-well plate. Choosing healthy cell is very important for enhancing transfection efficiency. The quantity of sirna (DNA) and DNA and the ratio between the sirna (DNA) and RNAi-Mate can be adjusted slightly within the recommended range. E. Suspension Cells Transfection Procedure F. DNA and sirna Cotransfecion 1. One day before transfection, incubate 4-5 x 10 4 cell into 24-well plate, add 0.5 ml culture medium containing FBS and antibiotics 2. Choose the appropriate cell quantity of primary incubation to make sure that cell fusion can reach 40-70%. 3. Dilute 1ug sirna (or 0.8 ug DNA) in 100 ul serum-free medium, add 3 ug RNAi-Mate reagent (when transfect DNA, 2.4 ug RNAi-Mate reagent is added) mix thoroughly, incubate at room temperature for 30 min in order to form sirna/rnai-mate (or DNA/RNAi-Mate) complex. 4. Add sirna/rnai-mate (or DNA/RNAi-Mate) complex into culture medium and mix up gently. 5. After incubating cells at 37 for 24 h-120 h, continue other steps for transfection. 1. On the day you start transfection, collect cells and centrificate, then resuspend in the FBS culture medium. 2. Dilute 1ug sirna (or 0.8 ug DNA) in 100 ul serum-free medium, add 3 ug RNAi-Mate reagent (when transfect DNA, 2.4 ug RNAi-Mate reagent is added), then add the above mixture into the wells of the 24-well plate. 3. Incubate at room temperature for 30 min in order to form sirna/rnai-mate (or DNA/RNAi-Mate) complex. 4. Add 400 ul cell suspension solution(cell quantity is determined by cell type and the time needed for analysis after transfection) 5. After incubating cells at 37 for 24 h-120 h, continue other steps for transfection. 1. One day before transfection, incubate 4-5 x 10 4 cell into 24-well plate, add 0.5 ml culture medium containing FBS and antibiotics 2. Choose the appropriate cell quantity of primary incubation to make sure that cell Page 5 of 6
fusion can reach 40-70 %. 3. Dilute 1ug sirna (or 0.8 ug DNA) in 100 ul serum-free medium, add 3 ug RNAi-Mate reagent (when transfect DNA, 2.4 ug RNAi-Mate reagent is added) mix thoroughly, incubate at room temperature for 30 min in order to form sirna/rnai-mate (or DNA/RNAi-Mate) complex. 4. Add sirna/rnai-mate (or DNA/RNAi-Mate) complex into culture medium and mix up gently. 5. After incubate cells at 37 for 24 h-120 h, continue other steps for transfection. G. Transduction In Vivo 1. Appropriate amount sirna and DNA is dissolved in RNAase-free sterile water, mix up gently, because volume of the injection is limited, we suggest high concentration sirna or DNA, generally DNA is 2 ug/ul, sirna is 10 ug /ul. 2. Mix up appropriate amount DNA, sirna or sirna\dna complex with RNAi-Mate. For example, in No.1 tube add 0.5 ul of DNA (1ug) and 0.5 ul of sirna (5 ug), in No.2 tube add 0.55 ul of RNAi-Mate (24 ug) and 0.45 ul of RNAase-free sterile water, the collect No.1 tube solution and add them into No.2 tube, incubate at room temperature for 30 min, in order to form sirna/rnai-mate (or DNA/RNAi-Mate) complex. 3. Preparation sirna/dna-rnai-mate complex can be used to transduct sirna, DNA sirna\dna in vivo. Page 6 of 6