INSTRUCTION MANUAL HiViTM Transfection Reagent

Transcription

1 Transfection reagents from labtech.com INSTRUCTION MANUAL HiViTM Transfection Reagent Labtech International Ltd 2 Birch House, Brambleside, Bellbrook Industrial Estate, Uckfield, TN22 1QQ Tel: Fax: sales@labtech.com Web:

2 TABLE OF CONTENTS Important User Information Section 1: Product Description Section 2: Shipping and Storage Section 3: Instructions before use Section 4: Protocols 4.1 Quick protocol for adherent cells 4.2 Protocol for suspension cells 4.3 Protocol for sirna 4.4 Protocol for sirna-dna co-transfection Section 5: Protocol Optimisation Section 6: Quality Control Section 7 : Troubleshooting

3 SECTION 1 Product Description HiVi TM Transfection Reagent is suitable for the transfection of all types of nucleic acid, providing exceptional transfection efficiency and transgene expression levels, whilst maintaining high cell viability. Part Code HIVI500 HIVI1000 HIVI5000 Description HiVi TM transfection reagent 500µl, tranfections per µg of DNA HiVi TM transfection reagent 1000µl, tranfections per µg of DNA HiVi TM transfection reagent 5 x 1000µl, tranfections per µg of DNA Benefits of HiVi: Exceptional cell viability and reproducible transfection efficiency even in sensitive cell lines Outstanding transgene expression levels Biodegradable breaks down with no adverse effect on the cell Transfects all types of nucleic acids into a variety of cell lines and primary cells. Ideal for High Throughput Screening Serum compatible Quick and easy to use protocol Can be used with Magnetic transfection technology SECTION 2 Shipping and Storage HiVi TM transfection reagent is shipped at room temperature. Upon receipt and for long term use, store at -20 o C as this minimizes the size of the liposomes which will lead to higher efficiency. HiVi TM reagents are however, stable for a minimum of 1 year at room temperature, +4 o C or -20 o C. The number of freeze/thaw cycles does not affect the efficiency of the reagent.

4 SECTION 3 Instructions before use Please ensure the HiVi TM protocol only is used to transfect with HiVi reagent. The instructions provided below were applied successfully with a number of cell lines, however optimal results may vary depending on the nucleic acid, cell type, size of the cell culture dishes and whether serum is used. The quantities and ratios of the individual components (DNA and HiVi reagent) may need to be adjusted to achieve the best transfection results. We therefore recommend that you optimise the various parameters as described in the Optimisation Protocol. As a starting point, we suggest 4µl of HiVi/1µg of DNA. As HiVi TM can be used with serum or in serum free conditions, it is possible to use your standard culture medium for the transfection but not while preparing the HiVi/DNA mix, this must be serum free. Cells should be healthy during their exponential growing phase. The presence of mycoplasma/fungi and other contaminants will affect the transfection efficiency. The proliferation rate is a critical parameter and optimal confluency will need to be adjusted according to the cells used. It is recommended to use cells that have been regularly passaged and to avoid cells that have been cultured for any length of time (>2 months). As a general rule, sirna transfection requires lower cell density than DNA transfection. Nucleic Acids need to be as pure as possible with low endotoxin levels. Avoid lengthy incubation time of the DNA/RNA solution before adding the HiVi reagent to prevent any degradation or surface adsorption. We have not noticed any significant effect of the use of/lack of antibiotics with the HiVi reagent. Whilst glass or polystyrene tubes can be used to prepare the DNA/reagent mix, we recommend polypropylene. For adherent cells, we recommend seeding or plating the cells the day before transfection. The suitable cell density will depend on the growth rate and condition of the cells. Cells should not be less than 60% confluent at the time of transfection. See suggested cell number in the table below. The recommended optimal plating density is dependent upon the time between transfection and transgene analysis. For a larger interval, a lower density is recommended, for a short interval, a higher density. T/C Dish Adherent Cell Number DNA Qty (µg) HiVi volume (µl) Dilu>on volume (µl) Transfec>on volume 96 well x x µl 24 well x x µl 12 well 1-2 x x 50 1ml 60mm dish 5-10 x x 150 4ml mm dish x x 250 8ml T75 flask x x ml Table 1: Cell number, DNA amount, HiVi TM volume and transfections conditions suggested For suspension cells, split the cells the day before transfection at a density of 2 to 5 x 10 5 cells/ml. It is possible to use the same protocols for stable transfection, however it is recommended to transfer cells to fresh media containing the required antibiotics after a minimum of 48hrs following transfection or 72hrs for suspension cells.

5 SECTION 4 Protocols 4. 1 Quick protocol for adherent cells The DNA and HiVi TM solutions should be at ambient temperature and gently vortexed prior to use (see suspension cells section 4.2). 1. Add 25 to 350µl of PBS or serum free media w/o antibiotics into two tubes 2. Add 0.25 to 20µg of DNA solution to one tube, making sure to add the DNA directly into the media, avoiding the sides of the tube. 3. Thaw the HiV TM i reagent at room temp. Add 1 to 80µl of reagent directly into the second tube, avoiding the sides as per point Add DNA solution to the HiVi TM reagent and mix by pipetting up and down 2/3 times. The diluted solutions should be combined within 5 minutes 5. Incubate for 15 to 20 minutes. Do not vortex or centrifuge. 6. Add the reagent mix to the cells in the serum containing culture media, drop by drop. Gently rock the plate to ensure a uniform distribution of the mixture. 7. Incubate at 37 o C in a CO2 incubator under standard conditions until ready to evaluate the transgene expression (24-72hrs). This will depend upon cell type and promoter activity. I. For some cells it may be necessary to replace old media with fresh media after 24hrs. II. For sensitive cells, 3-4hrs may be necessary Figure 1: Rapid protocol

6 4.2 Protocol for suspension cells The DNA and HiVi TM solutions should be at ambient temperature and be gently vortexed before use. For suspension cells, it is recommended to test several amounts of DNA with transfection reagent/dna ratio constant or several ratios with one amount of DNA. (Try 3 DNA quantities while keeping the DNA/HiVi ratio constant at 1/3). Please refer to the Optimisation Protocol later in the manual. 1. The day before transfection, split the cells at a density of 2 to 5 x 10 5 cells/ml so they are of the highest quality on the day of transfection. 2. Incubate overnight in serum containing media. 3. The day of transfection, prepare the HiVi/DNA mix as per the Adherent Cells protocol and incubate for minutes (no more than 30 minutes, do not vortex or centrifuge) 4. While the complexes are incubating, prepare the cells in serum free medium (or serum containing medium) and transfer the appropriate volume to the culture dish according to the table below. As a side note, serum free conditions generally lead to higher transfection efficiency. 5. Add the reagent mix to the cells, drop by drop and all over the well. Mix the complex with the cells by pipetting the media up and down, a number of times. This is to ensure complete contact of the complex with cells that are prone to clumping. It is very important to promote contact of the cells with the complex at this stage. The pipetting disrupts the clumping and produces a single cell suspension that increases transfection efficiency. 6. Incubate 3 to 6hrs in serum free medium at 37 o C under 5% CO2. If the media contains serum, go to step Add more culture medium containing 20% serum (same volume as the transfection volume) 8. Incubate at 37 o C in a CO2 under standard conditions until ready to evaluate transgene expression (24-72hrs). This will depend upon cell type and promoter activity. I. For some cells it may be necessary to replace old media with fresh media after 24hrs. II. It is possible to just add fresh growth media, bearing in mind the addition of the transfection mix dilutes supplements in your standard culture media. T/C Dish Suspension Cell Number DNA Qty (µg) HiVi volume (µl) Transfec>on volume 96 well x µl 24 well 2-4 x µl 6 well x ml 60mm dish 5 x ml Table 2: Transfection conditions suggested for suspension cells Important notes for achieving optimum transfection results: Whilst transfection is possible in the presence of serum, optimum transfections are found when performed in serum free conditions. It is important to promote as much contact between the cells and the reagent mix. This is possible by: I. Concentrate your cells. Whilst preparing the reagent mix, spin down the cells, re-suspend at 10x10 6 cells/ml in serum free media and transfer the appropriate cell number to the wells as per the above table and carry on with the protocol. II. Promote contact by centrifugation. Centrifuge the plate after having mixed the cells with the transfection complexes for a couple of minutes between rpm

7 4.3 Protocol for sirna 1. The day before transfection, prepare the cells as described earlier. sirna generally requires lower cell density than DNA transfection. The choice of optimal plating density will depend on the planned time between transfection and gene knockdown analysis. For a large interval, we recommend lower density however for a short interval, higher density. 2. Ensure the sirna and hivi TM reagent is at ambient temperature and gently vortexed before use and mixed together within 5 minutes. 3. Dilute the sirna solution in 25 or 50µl of PBS or serum and antibiotic free culture media. It is recommended to start with a final sirna concentration of 50nM. See table 3 4. Dilute HiVi TM reagent in 25 or 50µl of PBS or serum and antibiotic free culture media. For very small volumes, pre-dilute HiVi TM in deionized water. See table 4 5. Mix the two solutions by gently pipetting up and down in the mixture. 6. Incubate for 20 minutes at room temperature. Do not vortex. 7. Add the mixture drop by drop directly onto the cells. Please see the table 1 for the total transfection volume per well. 8. Incubate the cells at 37 o C in a CO2 incubator under standard conditions until ready to evaluate gene silencing. The time will depend on the sirna amount, gene targeted and the cell type. Assays can be monitored 24 to 96hrs post transfection RNA analysis, 24hrs, 48-72hrs for protein knockdown analysis. I. For some cells it may be necessary to replace old media with fresh media after 24hrs. II. For sensitive cells, 3-4hrs after transfection or 24hrs after incubation with fresh media may be necessary Important notes for achieving optimum transfection results: Ensure serum free conditions are used when preparing the reagent/sirna mix. Use fresh media as some older media could change ph and could influence the assay. Do not incubate the diluted sirna for too long in the serum free media. It is advised to firstly prepare the transfection reagent, dilute the sirna and quickly transfer the diluted sirna into the HiVi TM tube. Start with 50nM sirna concentration and test four amounts of HiVi TM reagent. Gene silencing is dependent on your protein half-life and consequently it will be good to analyse your protein expression by Western at 48hrs, 72hrs and 96hrs Treating your cells twice with 25nM sirna rather than once with 50nM can significantly enhance sirna effects. Day 1, incubate cells with 25nM sirna and HiVi TM. Day 2, change media and repeat with 25nM sirna and HiVi TM Culture vessel 96 well 24 well 12 well 6 well Dilu>on serum- free medium or PBS 25µl 25 µl 25 µl 25 µl Amount of sirna (1µM stock)* Final sirna concentra>on µl ng µl ng µl ng µl ng 20nM nM Table 3: Suggested dilution procedure and amount of sirna to test

8 Culture vessel 96 well 24 well 12 well 6 well Dilu>on serum- free medium or PBS 25µl 25 µl 25 µl 25 µl Amount of HiVi transfec>on Reagent Final sirna concentra>on 20nM 0.3 µl 1 µl 2 µl 4 µl 50nM 0.5 µl 2 µl 4 µl 8 µl Table 4: Suggested amount of HiVi reagent per nm of sirna used 4.4 Protocol for sirna-dna co-transfection HiVi TM Transfection Reagent is also suitable for co-transfection of sirna and plasmid DNA. Please remember the following before starting the transfection process: The choice of optimal plating density will depend on the planned time between transfection and gene knockdown analysis. For a large interval, we recommend lower density however for a short interval, higher density. Always use a volume of reagent that is at least twice the quantity of nucleic acid (2µl per µg of DNA) For co-transfection of several plasmid DNA, mix the same amount of each plasmid and transfect as described above in the Adherent Cells or Suspension Cells protocol. E.g. if you have 2 plasmids, mix 2.5µg of each, mix the 5µg of plasmid with at least 10µl of HiVi TM reagent (20µl is the recommended volume) The DNA, sirna and HiVi TM mix should be at ambient temperature and vortexed gently before use. 1. Plate the cells as described in Adherent Cells or Suspension Cells protocol. 2. Prepare the reagent/plasmid mix. There are 2 options: Option 1) Prepare the HiVi/DNA mix and HiVi sirna mix in 2 different tubes Prepare the HiVi/DNA mix as per the Adherent cells Protocol, incubate for 15 minutes. Prepare the HiVi/siRNA mix as per the sirna protocol, incubate for 15 minutes. Combine the above solutions, mix gently by pipetting up and down (do not vortex) and add directly to the cells. Option 2) Prepare the HiVi/Nucleic acid mix by pooling the DNA and sirna together and then add the transfection reagent. a. Dilute the sirna and DNA together in 1 tube as described above b. Prepare the HiVi TM solution in a separate tube. Add a sufficient amount of HiVi TM for both DNA and sirna. To start, try HiVi/total Nucleic Acid (DNA and sirna) ration of 5µl HiVi TM per µg of total nucleic acid. c. Mix the 2 solutions by carefully pipetting up and down, incubate for minutes at room temperature do not vortex. 3.Add the mix, drop by drop onto the cells growing in the serum-containing media and homogenise by gently rocking the plate side to side to ensure the mixture is fully distributed around the well. 4.Incubate the cells at 37 o C in a CO2 incubator under standard conditions until ready to evaluate gene knockdown. This will depend on the amount of sirna, the targeted gene and the cell type. Assays can be monitored 24 to 96hrs post transfection. We recommend for RNA analysis, 24hrs, 48-72hrs for protein knockdown analysis. i. For some cells it may be necessary to replace old media with fresh media after 24hrs ii. For sensitive cells, 3-4hrs after transfection or 24hrs after incubation with fresh media may be necessary It is also possible to transfect the nucleic acid at different times rather than simultaneously. Cells can be transfected with sirna first and 4 to 24hrs later transfected with DNA. Follow the procedures as detailed above for DNA and sirna transfection. A media change can also be performed before the DNA transfection.

9 SECTION 5 Optimisation Protocol Whilst high transfection efficiencies can be achieved in a wide range of cell types with the rapid protocols, some optimisation may be needed to achieve maximum efficiency in certain cells. Optimisation of the transfection protocol is recommended for each combination of plasmid and cell line used in order to achieve the best out of the reagent. Several parameters can be optimised. Ratio of HiVi TM reagent to nucleic acid (DNA/siRNA/RNA) Dose of nucleic acid used Cell type and cell density Culture medium composition (with or without serum) and reagent/nucleic acid mix. Incubation time It is recommended to optimise one parameter at a time whilst keeping the others constant. Two most critical variables are the ratio of HiVi TM reagent to DNA (sirna) and the quantity of DNA (sirna concentration) 1. HiVi TM Reagent/DNA ratio HiVi TM Reagent must be used in a larger quantity compared to DNA however the optimum ratio will depend on the cell line and vessel used. To optimise, firstly maintain a fixed quantity of DNA (as per the recommendations for the particular culture dish used), then vary the amount of HiVi TM reagent over the suggested range as per table 5. It is recommended to test ratios from 1 to 6µl of HiVi TM reagent for each µg of DNA. T/C Dish DNA Qty (µg) HiVi volume (µl) HiVi volume (µl) proposed interval 96 well well well mm dish mm dish T75 flask Table 5: Suggested range HiVi reagent for optimisation 2. HiVi TM Reagent/siRNA ratio Use a fixed amount of sirna and vary the amount of HiVi TM reagent as detailed in table 6. The reagents can be pre-diluted in deionized water and the aliquots can be incubated with sirna. Diluted HiVi TM solution must be freshly prepared. Culture vessel 96 well 24 well 12 well 6 well Final transfec>on volume 200µl 500µl 1ml 2ml Amount of HiVi transfec>on Reagent Final sirna concentra>on 25nM µl µl µl µl 50nM µl µl µl µl Table 6: Suggested amount of HiVi TM reagent per nm of sirna used.

10 3. Quantity of DNA or sirna It is possible to achieve optimum transfection efficiency by increasing the amount of DNA or concentration of sirna. However, too high an amount can lead to over expression or lysis of the cells. These effects vary with the number of cells present therefore, it is important to keep the number of cells and incubation time constant during the optimisation process. So, following the determination of HiVi reagent/dna ration, or HiVi TM reagent /sirna ratios, in order to figure out the best amount of DNA or concentration of sirna, maintain a fixed ratio of the HiVi TM reagent to DNA/siRNA and vary the DNA quantity over the suggested range. See below. Following this, culture medium compositions, cell number and incubation times can also be optimised. T/C dish DNA quan>ty (µg) Transfec>on volume 96 well µl 24 well µl 12 well 1-4 1ml 60mm dish ml T- 75 flask ml Table 7: Suggested range of DNA amounts for optimisation 3. HiVi TM Reagent/Nucleic Acid media mix Research has shown that the use of PBS rather than serum and antibiotic free media to prepare the DNA/ RNA/siRNA and HiVi TM solution mix leads to more reproducible transfections and in some cases higher efficiency particularly those with lower volumes of transfection reagent. 4. Cell Number Depending on the cells used, it will be necessary to adjust the optimal confluency. By using the information obtained from the optimised ratio and DNA amount, vary the number of cells to be assayed. Please remember, the addition of the transfection mix to freshly seeded cells can result in a considerable increase in transfection efficiency. For stable transfection it is possible to seed cells with a lower density and, because of the efficiency of HiVi TM Transfection reagent, it is also possible to lower the quantity of DNA. Transfer cells to fresh medium containing the appropriate antibiotics, 48 to 72 hours after transfection. It is important to wait at least 48 hours before introducing the cells to selection media, however for some cells this could be as long as 4-5 days. 5. Effects of Serum/Transfection Volume: Almost all cell lines transfected with HiVi TM showed superior results if serum is present during transfection. Some cell lines may differ and transfection efficiency can be increased in serum free or reduced serum conditions. Please remember that serum is not to be present during the formation of the transfection mix as the serum will inhibit the formation. Transfection efficiency is obtained during the initial 3-4 hours of incubation so the cells can be kept in serum free or reduced serum conditions during this time. If you generally use serum free media, replace it with a media containing serum, or add the required amount of serum to the wells, after this period. If required, to increase the efficiency of transfection, you can reduce the transfection volume. 6. Incubation time: The optimal time range between transfection and evaluating gene activity varies with cell lines, promoter activity, expression product etc. The transfection efficiency can be monitored after hours by analysing the gene product. Reporter genes can be used to quantitatively measure gene expression.

11 SECTION 6 Quality Control Each batch of HiVi TM Transfection reagent is tested for purity, sterility and biological activity as per the table below. Test Purity Sterility Biological Ac>vity Method Silica gel TLC assays, every compound shall have a single spot Thioglycolate assay. Any sign of fungus and bacterial contamina>on is monitored over a 7 day period. Transfec>on efficacies on NIH- 3T3 and COS7 cells. Every batch shall have >80% ac>vity when compared with the reference batch.

12 SECTION 7 Troubleshooting Problem Low Transfec>on efficiency Checks/Recommenda>ons HiVi TM Reagent/nucleic acid ra>o. Op>mise the reagent/nucleic acid ra>o by using a fixed amount of DNA (µg) or sirna (nm) and vary the amount of HiVi TM reagent from 2 >mes less up to 3 >mes more than the recommended amount detailed in the earlier protocols. DNA Amount Use different amounts of DNA with the recommended or op>mised reagent/dna ra>o Cell density The op>mal cell density should range from 50% to 70% (true confluency, corresponding to 90% visual confluency) as non- op>mal cell density at the >me of transfec>on can lead to insufficient uptake. The most favourable cell density may vary according to the cell type. Quality of DNA or sirna The nucleic acid should be as pure as possible, free of any contaminants (protein, phenol etc) and a low endotoxin. Ensure all materials are nuclease free. Type of promoter Ensure that the DNA promoter can be recognised by the cells that are to be transfected. Other cells or a viral driven reporter gene expression can be used as a control. Cell condi>on Cells that have been in culture for a long >me may become resistant to transfec>on. Freshly thawed cells recently passaged are recommended. Cells should be healthy and assay during their exponen>al growing phase. Any contaminants present will alter the transfec>on efficiency Media For preparing DNA/reagent mix It is essen>al that serum- free media or buffer is used to prepare the DNA/reagent mix. Avoid contact of the reagent/nucleic acid solu>on with the plas>c surface. Media For transfec>on For some cells, transfec>on efficiency can be increased without serum or under reduced serum condi>ons, therefore, transfect these cells in serum free medium during the first 4hrs of incuba>on Incuba>on >me and transfec>on volume Transfec>on efficiency can be monitored between 24 and 96 hours by analysing the gene product. If efficiency is low, try reducing the transfec>on volume for the first 24 hours Old reagent/dna mix It is essen>al to use freshly prepared reagent/dna mix, complexes prepared and stored longer than 1 hour can be aggregated. Transgene detec>on assay Ensure your post- transfec>on assay is properly set up and includes a posi>ve control. Reagent temperature All reagents should have an ambient temperature and be vortexed prior to use. Storage temperature Transfec>on efficiency can slowly decrease if kept at +4 o C for more than 1 week. Store at - 20 o C to recover ini>al efficiency.

13 Cellular Toxicity Unhealthy cells Check for contamina>on Use a new batch of cells Ensure correct media condi>on (ph, type of media etc) Cells are too confluent/cell density is too low Check equipment/materials Transgene product is toxic Use suitable controls for detec>on sirna/dna quality Use pure nucleic acid, ensure contaminant and endotoxin free. Too high a concentra>on Decrease amount of nucleic acid/reagent mix added to the cells Aggrega>on of reagent mix can cause some toxicity, prepare fresh mix and adjust ra>os where necessary Incuba>on >me Reduce the incuba>on >me by replacing the transfec>on media with fresh media between 4 and 24hours. Key gene silencing If the targeted gene is essen>al for cell survival or if a key gene is non- specifically silenced by the sirna, this can lead to cell death. None, or limited gene silencing sirna design Make sure a validated sirna sequence is used. If this is not possible, assay your sequence in an easy to transfect cell line in order to validate it. sirna concentra>on use a higher amount of sirna if necessary Incuba>on >me Perform a >me course experiment to set up the op>mal incuba>on >me as gene silencing is dependent upon gene expression and the protein turnover rate Medium used for preparing transfec>on reagent/sirna mix Serum free media or buffer must be used during prepara>on of the reagent/nucleic acid mix. Old reagent/sirna mix Use freshly prepared reagent/sirna mix Labtech International Ltd 2 Birch House, Brambleside, Bellbrook Industrial Estate, Uckfield, TN22 1QQ, UK. Tel: Fax: sales@labtech.com Web: