sirna / mirna transfection KIT GenomONE - Si Instruction Manual (Ver.3.1)

Transcription

1 sirna / mirna transfection KIT GenomONE - Si Instruction Manual (Ver.3.1) 1. Outline :Principles and outline of transfection :Specifications sirna/mirna introduction into cells(in vitro) :Protocol 1 Basic protocol :Protocol 2 Centrifugation protocol :Protocol 3 Basic protocol + centrifugal treatment Rapid transfection for multiple types of and numerous samples(high throughput screening:hts) :Protocol 4 HTS-RT method(reverse Transfection method) :Protocol 5 HTS-FT method(forward Transfection method) sirna/mirna introduction into laboratory animals (in vivo) :Protocol 6 in vivo Method Precautions for use Outline 1-1:Principles and outline of transfection sirna/mirna is incorporated into the HVJ envelope (HVJ-E) to obtain the HVJ-E vector, which is then introduced into target cells/tissues using the membrane fusion activity of fusion (F) proteins. With the basic protocol (1), transfection can be completed in 5 steps (5-10 minutes). (1) (2) (3) (4) (5) HVJ-E Suspension Reagent D sirna mirna! This product is for use in laboratory research. It has not been approved for in vitro or in vivo use for the diagnosis or treatment of a patient and the seller advises against any such use.! This package insert describes standard methods to be used with GenomONE -Si for transfection with sirna,mirna, etc. The methods described here yield reasonable efficiency of transfection, though optimal conditions of transfection can vary depending on cell type. It is advisable to optimize the conditions of transfection, referring to the precautions listed in this package insert. 1 Reagent E addition

2 1-2:Specifications 2 Refrigerated at 2-8C. Product name Cat. # Freeze-dried HVJ-E (inactivated HVJ) Reagent D Reagent E Buffer 0.26 ml /vial (when reconstituted) 0.5mL/vial 4.0mL/vial 6.5mL/vial GS GenomONE-Si GS GS GS [Role of each reagent] Reagent D (reagent for incorporation):increases permeability across the HVJ-E membrane. Reagent E (enhancer for introduction):a positively-charged peptide which increases affinity between the molecule-bearing HVJ-E (HVJ-E vector) and the cell (or tissue) and thus increases the efficiency of transfection. [Frequency of use] Cat. # HVJ-E (vial) Frequency of use (wells) 6-well plate 24-well plate 96-well plate 384-well plate GS ,000 5,000 GS ,600 8,000 20,000 GS ,600 6,400 32,000 80,000 GS ,000 16,000 80, ,000 [Storage, stability, and quality assurance] Quality assured period of freeze-dried HVJ-E: See the aluminum package for HVJ-E. Because the activity of freeze-dried HVJ-E can be reduced by exposure to high temperature or high relative humidity, refrigerated storage with sealing in an aluminum package is required. HVJ-E suspension prepared with buffer: For continuous use, store in a refrigerator (2-8 o C) for up to two weeks. For extended storage, freeze in working aliquots at -80 o C for up to 3 months. Thawing after freezing is possible only once. [Quality and safety] Although HVJ-E uses HVJ (Sendai virus) as a raw material, the genomic RNA of HVJ has been completely inactivated by drug treatment*. The HVJ-E will not proliferate or exhibit pathogenic effects in humans or animals. *Reference: Kaneda, Y. et al.: "Non-Viral Vectors for Gene Therapy, Advances in Genetics, Vol. 53, pp , Ed. Huang Leaf, Hung Mien-Chie, Wagner Ernst, Academic Press (2005). Related article: Prior, C. et al.: BioPharm, (Oct. 1996)! HVJ-E retains membrane-fusion activity. Therefore, to prevent inhalation, attachment, unintended swallowing, or spread to eyes or nose of the HVJ-E particles, the product must be manipulated within a safety cabinet, wearing appropriate clothing (laboratory overalls) and protective items (plastic or latex gloves, mask, protective eyeglasses, etc.). Inactivation of HVJ has been confirmed for each lot by the viral proliferative potential rule-out test, using cultured cells and fertilized chicken eggs. Absence of contamination by bacteria and fungi has been confirmed by sterility testing.! Absence of contamination by all microorganisms cannot be guaranteed and appropriate procedures must be followed when using this product. Endotoxin level has been confirmed to be less than 2.5 EU/mL (Limulus amebocyte lysate gel clot assay). Expression of the gene introduced in cultured cells (BHK-21; ATCC CCL-10) in the presence of serum has been confirmed.

3 What is HVJ Envelope(HVJ-E)? HN protein F protein inactivation purification HVJ (Sendai virus) HVJ Envelope (HVJ-E) (Inactivated Sendai virus) HVJ Envelope is a purified product prepared through complete inactivation of Sendai virus (HVJ: Hemagglutinating Virus of Japan). It is a vesicle in which only the cell membrane-fusing capability of the envelope protein of Sendai virus is retained. The genomic RNA of the Sendai virus contained in HVJ-E has been inactivated completely and has neither infective nor proliferative potentials in humans or experimental animals. HVJ-E can be used safely at ordinary laboratories, without requiring any special operations or facilities. Conventional non-viral vectors, including cationic lipids are incorporated into cells through endocytosis which results in degradation of most parts of the transferred DNA by lysosomes 2 sirna/mirna introduction into cells (in vitro) 2-1. Protocol 1: Basic protocol Applicable to both Adherent cells and Suspension cells. Initial assessment should use Protocol (1). Preparation of HVJ-E suspension (reconstitution of freeze-dried HVJ-E) Ice-cooled buffer (0.26 ml) is added to a tube containing freeze-dried HVJ-E. The mixture is pipetted gently, with care taken to avoid bubbling. A homogeneous suspension is thus prepared and stored refrigerated. Immediately before use, HVJ-E suspension, other reagents, and tubes should be cooled adequately in an ice bath. The vector is then prepared in the following steps. See page 2 for storage method. Precautions for Use Please use the HVJ-E suspension, the sirna/mirna solution, and the Reagents D and E, which have been cooled in ice. Recommended sirna (oligo-type) concentration: 10μM(2-50μM)(21nt sirna:10μm=10pmol/μl = ~0.14μg/μL) Protocol 1: Basic protocol Step Amount of reagent (6-well plate /well) (1) HVJ-E suspension taken into a micro-test tube HVJ-E suspension: 2.5μL (2) Combination with Reagent D and agitation (tapping) Reagent D:0.5μL (3) Combination with sirna /mirna solution and agitation (tapping) sirna /mirna solution: 10μL (4) Combination with Reagent E and agitation (tapping) Reagent E: 5μL (5) Add the suspension that has been prepared in Step (4) to the cell culture fluid in the wells, and incubate it under the conditions at 37ºC/5% CO2. 1 (The medium is reviewed hours later, as needed.) 3 HVJ-E vector suspension (1)+(2)+(3)+(4): 18μL Steps (1) through (4) should be performed on ice (To avoid reduction of HVJ-E activity due to temperature rise). The amount of Reagent D added is equal to 1/5 of the HVJ-E suspension [Step (2)]. The transfection of sirna / mirna into cultured cells is possible even in the presence of serum. 1 :Please monitor gene silencing efficiency at an appropriate time (e.g., 6 to 72 hours after transfection, although it depends on the experimental system).

4 Amount of reagent for each plate Plate size HVJ-E suspension(1) Reagent D (2) sirna/mirna solution (3) Reagent E (4) Amount of HVJ-E vector to be treated (5) 6-well 2.5μL 0.5μL 10μL 5μL 18μL/well 1 well 24-well 2.5μL 0.5μL 10μL 5μL 4.5μL/well 4 wells 96-well 2.5μL 0.5μL 10μL 5μL 0.9μL/well 20 wells [Recommended cell density for each well plate size (Adherent cells)] Plate size Cell density (upon inoculation onto the well plate 2 ) 6-well 24-well 96-well 0.4~ cells/2.0ml of medium/well 1.0~ cells/0.5ml of medium/well 0.2~ cells/0.1ml of medium/well 2 Used for transfection under conditions of one-day culture and 50-80% confluency. [Recommended cell density for each well plate size (Suspension cells)] (The recommended cell density is around 10-fold the count given below in case of primary cultures of primary immune cells.) Plate size 6-well 24-well 96-well Cell density (upon inoculation onto the well plate*) 0.8~ cells/2.0ml of medium/well 0.2~ cells/0.5ml of medium/well 0.4~ cells/0.1ml of medium/well [Troubleshooting] Low efficiency of transfection Efficiency may be increased by the following measures: The concentration of sirna/mirna solution is elevated. Increase the amount of HVJ-E suspension. Evaluation is made with Protocol 3. High cytotoxicity If any sign of cytotoxicity is noted, cytotoxicity may be reduced by the following measures: Use the HVJ-E suspension and the reagents D and E after dilution with a buffer solution within a range of about 4-fold dilution. Evaluation is made with Protocol 2, Protocol 2: Centrifugation protocol If Protocol 1 results in high cytotoxicity, Protocol 2 should be used to improve the situation. Protocol 2 requires the addition and mixing of Reagent D with the HVJ-E suspension, which is then centrifuged, and the resultant sediment is resuspended in Buffer. Preparation of HVJ-E suspension (reconstitution of freeze-dried HVJ-E) Ice-cooled buffer (0.26 ml) is added to a tube containing freeze-dried HVJ-E. The mixture is pipetted gently, with care taken to avoid bubbling. A homogeneous suspension is thus prepared and stored refrigerated. Immediately before use, HVJ-E suspension, other reagents, and tubes should be cooled adequately in an ice bath. The vector is then prepared in the following steps. See page 2 for storage method. Recommended sirna (oligo-type) concentration: 10μM(2~50μM)(21nt sirna:10μm=10pmol/μl = ~0.14μg/μL) Precautions for Use Please use the HVJ-E suspension, the sirna/mirna solution, the Buffer, and the reagents D and E, which have been cooled in ice. 4

5 Protocol 2: Centrifugation protocol Step Amount of reagent (6-well plate /well) (1) HVJ-E suspension taken into a micro-test tube HVJ-E suspension: 2.5μL (2) Combination with Reagent D and agitation (tapping) Reagent D: 0.5μL (3) Centrifugation at 10,000 g(10,000-12,000rpm) for 5 minutes at 4 C. (4) Supernatant is discarded.sediment suspended in the buffer Buffer:3.0μL (5) Combination with sirna /mirna solution and agitation (tapping) sirna/mirnasolution:10μl (6) Combination with Reagent E and agitation (tapping) Reagent E:5μL Add the suspension that has been prepared in Step (6) to the cell (7) culture fluid in the wells, and incubate it under the conditions at HVJ-E vector suspension 37ºC/5% CO2. 3 (The medium is reviewed hours later, as [(4)+(5)+(6)]: 18μL needed.) Steps (1) through (6) should be performed on ice (To avoid reduction of HVJ-E activity due to temperature rise). The amount of Reagent D added is equal to 1/5 of the HVJ-E suspension [Step (2)]. The transfection of sirna / mirna into cultured cells is possible even in the presence of serum. 3 :Please monitor gene silencing efficiency at an appropriate time (e.g., 6 to 72 hours after transfection, although it depends on the experimental system). Amount of reagent for each plate Plate size HVJ-E suspension(1) Reagent D (2) Buffer (4) sirna/mirna solution(5) Reagent E (6) Amount of HVJ-E vector to be treated (7) 6-well 2.5μL 0.5μL 3.0μL 10μL 5μL 18μL/well 1 well 24-well 2.5μL 0.5μL 3.0μL 10μL 5μL 4.5μL/well 4 wells 96-well 2.5μL 0.5μL 3.0μL 10μL 5μL 0.9μL/well 20 wells [Cell density] For cell density of adherent and suspension cells at the time of transfection, refer to page 3 (P.5 6). [Troubleshooting] Low efficiency of transfection Efficiency may be increased by the following measures: The concentration of sirna/mirna solution is elevated. Increase the amount of HVJ-E suspension. Evaluation is made with Protocol 3. High cytotoxicity If any sign of cytotoxicity is noted, cytotoxicity may be reduced by the following measures: Use the HVJ-E suspension and the reagents D and E after dilution with a buffer solution within a range of about 4-fold dilution Evaluation is made with Protocol Protocol 3: Basic protocol + centrifugal treatment When applied to suspension cells, high cytotoxicity or low introduction efficacy with Protocol 1, 2 may be improved with the use of Protocol 3 which involves centrifugation after the sirna/mirna-including HVJ-E vector suspension is combined with cells. Preparation of HVJ-E suspension (reconstitution of freeze-dried HVJ-E) Ice-cooled buffer (0.26 ml) is added to a tube containing freeze-dried HVJ-E. The mixture is pipetted gently, with care taken to avoid bubbling. A homogeneous suspension is thus prepared and stored refrigerated. Immediately before use, HVJ-E suspension, other reagents, and tubes should be cooled adequately in an ice bath. The vector is then prepared in the following steps. See page 2 for storage method. Recommended sirna (oligo-type) concentration: 10μM(2~50μM)(21nt sirna:10μm=10pmol/μl=~0.14μg/μl) Precautions for Use Please use the HVJ-E suspension, the sirna/mirna solution, and the Reagents D and E, which have been cooled in ice 5

6 Protocol 3: Basic protocol + centrifugal treatment Steps (1) through (4) are identical to those of Protocol 1. Step Amount of reagent (6-well plate /well) (1) HVJ-E suspension taken into a micro-test tube HVJ-E suspension: 2.5μL (2) Combination with Reagent D and agitation (tapping) Reagent D: 0.5μL (3) Combination with sirna/mirna solution and agitation (tapping) sirna /mirna solution: 10μL (4) Combination with Reagent E and agitation (tapping) Reagent E: 5μL (5) HVJ-E vector suspension that has been prepared in Step (4) is combined with the cells suspended in medium (0.5 ml) in a tube. (6) Centrifugation at 5,000 g for 10 minutes at 4 C. 4 (7) The supernatant is discarded. The cells are re-suspended in 2.0 ml medium and transferred to a well plate for incubation at 37 C under 5%CO2. 5 HVJ-E vector suspension: 18μL cell: 0.5mL Medium for re-suspension:2.0 ml Dispense depending on plate size. Steps (1) through (4) should be performed on ice (To avoid reduction of HVJ-E activity due to temperature rise). The amount of Reagent D added is equal to 1/5 of the HVJ-E suspension [Step (2)]. The transfection of sirna / mirna into cultured cells is possible even in the presence of serum. 4 :The conditions of centrifugation (rate of rotation, temperature, and duration) may be adjusted within the range not causing cell damage.[step(6)] 5 :Please monitor gene silencing efficiency at an appropriate time (e.g., 6 to 72 hours after transfection, although it depends on the experimental system). [Dispensed volume, cell density and assay frequency depending on the suspension cell plate size] Centrifugation Medium for Inoculation onto the well plate Plate [Step(5)] re-suspension [Step(7)] assay size Cell density / tube [Step(7)] / tube Medium volume/well Cell density /well 6 (wells) 6-well 2.0mL cells cells 24-well 2.0mL 0.5mL cells 4 / 0.5mL of medium 96-well 0.1mL cells 20 6 :The cell density/well is identical to that of Protocol 1, 2. [Dispensed volume, cell density and assay frequency for Adherent cells (suspension cells after detachment)] Centrifugation Medium for Inoculation onto the well plate Plate [Step(5)] re-suspension [Step(7)] assay size Cell density / tube [Step(7)] / tube Medium volume/well Cell density /well 7 (wells) 6-well 2.0mL cells cells 24-well 2.0mL 0.5mL cells 4 /0.5mL of medium 96-well 0.1mL cells 20 7 :Cell density /well is twice that of Protocol 1. Transfection of suspension cells by using Protocol 3 6

7 [Troubleshooting] Low efficiency of transfection Efficiency may be increased by the following measures: The concentration of sirna/mirna solution is elevated. Increase the amount of HVJ-E suspension. High cytotoxicity If any sign of cytotoxicity is noted, cytotoxicity may be reduced by the following measures: Use the HVJ-E suspension and the Reagents D and E after dilution with a buffer solution within a range of about 4-fold dilution 3.Rapid transfection for multiple types of and numerous samples (High throughput screening:hts) 3-1. Protocol 4: HTS-RT method (Reverse Transfection method) After preparing the HVJ-E vector suspension (sirna/mirna is incorporated into HVJ-E) in a cell culture plate, the suspension can be used for the reverse transfection (RT) method (HTS-RT method), in which cells are added to the plate. Preparation of HVJ-E suspension (reconstitution of freeze-dried HVJ-E) The tube containing freeze-dried HVJ-E is combined with ice-cooled buffer (0.26 ml). The mixture is gently pipetted, with care taken to avoid bubbling. A homogeneous suspension is thus prepared. The HVJ-E suspension gradually loses activity if the temperature is above 8 C. The suspension should be immediately stored in an ice-cooled bath or in a refrigerator (2-8 C). See page 2 for storage method. Materials to be prepared separately for the HTS protocol: In the case of a shortage in the buffer solution, please use phosphate buffered saline (PBS: free from Ca 2+ and Mg 2+ ). Please consistently use a plate with non-treated surfaces for transfection into suspension cells. Recommended sirna (oligo-type) concentration:1μm(0.05-4μm) (21nt sirna:1μm=1pmol/μl= ~0.014μg/μL) Precautions for Use Please use the HVJ-E suspension, the sirna/mirna solution, Buffer, and the Reagents D and E, which have been cooled in ice Steps (1) through (4) should be performed on ice (To avoid reduction of HVJ-E activity due to temperature rise). When a tissue culture treated plate is used, the HVJ-E is adhered to the plate surface; therefore, please consistently use a plate with non-treated surfaces for suspension cells. However, for adherent cells, a decrease in growth is occasionally found by using a plate with non-treated surfaces depending on cell species; therefore, it is recommended to use a plate after examining beforehand whether optimal results can be obtained by the use of a certain type of plate, tissue culture treated or non-treated. Protocol 4: HTS-RT method (in a case of preparation of cells at a cell count corresponding to that in 100 wells of a 96-well plate) Step 7 Amount of reagent (1) HVJ-E suspension taken into a micro-test tube HVJ-E suspension: 12.5μL (2) Combination with Reagent D and agitation (tapping) Reagent D:2.5μL (3) Centrifugation at 10,000 g(10,000-12,000rpm) for 5 minutes at 4 C. Supernatant is discarded. (4) Sediment suspended in the buffer (After pipetting 100 μl 10 to 20 times, add 400 μl, then pipet once. 8 ) Buffer: 500μL (5) Add the sirna or mirna solution to each well in the 96-well plate. sirna/mirna solution:5μl (6) Add the suspension prepared in Step (4) to each well (pipetting 1 to 3 Suspension prepared in times), and then mix. Step (4):5μL (7) Add the reagent E that has been diluted with the buffer solution by 20-fold to each well, and then mix. 9 (pipetting 1 to 3 times) 1/20 Reagent E:5μL (8) Add the cell fluid prepared beforehand to each well, and incubate it under the conditions at 37ºC/5% CO2. (The medium is reviewed hours later, as needed.). 10 Cell fluid: 100μL Steps (1) through (4) should be performed on ice (To avoid reduction of HVJ-E activity due to temperature rise).

8 The transfection of sirna / mirna into cultured cells is possible even in the presence of serum. When in a state of Step (4), the HVJ-E suspension can be stored under refrigeration (4ºC) for 24 hours. In a case where the procedures (5) to (7) are carried out at room temperature using automatic dispensing equipment, etc., please consistently use each solution that has been cooled in ice, and carry out the procedures in each step promptly to avoid deterioration of the activity of the HVJ-E due to an increase in temperature. The amount of Reagent D added is equal to 1/5 of the HVJ-E suspension [Step (2)]. 8 :If the bottom part of the tube is held, such an action may lead to inactivation of the HVJ-E due to an increase in pellet temperature. Therefore, please pipet the sample by holding the top part of the tube. 9 :Please adequately mix by pipetting, etc., which can affect transfection efficiency. 10 :Please monitor gene silencing efficiency at an appropriate time (e.g., 6 to 72 hours after transfection, although it depends on the experimental system). [Amount of reagent for each well plate size] Plate size Reverse Transfection (RT) sirna/mirna solution (5) HVJ-E suspension (6) 1/20 Reagent E (7) 96-well 5μL 5μL 5μL 384-well 2μL 2μL 2μL [Recommended cell density for each well plate size (Suspension cells)] (The recommended cell count is around 10-fold the count given below in case of primary cultures of primary immuno cells.) Plate size 96-well 384-well Cell density (upon inoculation onto the well plate) 0.4~ cells/0.1ml of medium/well 0.16~ cells/0.04ml of medium/well [Troubleshooting] Low efficiency of transfection Efficiency may be increased by the following measures: The concentration of sirna/mirna solution is elevated. Increase the amount of HVJ-E suspension. Adequately mix the cells with the HVJ-E vector suspension by pipetting, etc., in Step (8). The use of small-diameter wells such as those of a 384-well plate makes it difficult to thoroughly mix the cells from Step (8) with the HVJ-E vector suspension, leading to reduced transfection efficiency. Ensure that the cells are well-mixed with the HVJ-E vector suspension. If thorough mixing is difficult to achieve, centrifuging at 1500 g for 5 min at 25ºC allows sufficient contact between the cells and the HVJ-E vector, thereby preventing a decrease in transfection efficiency. Depending on the type of plate with treated surfaces, transfection efficiency may likely be affected; therefore, in such a case please examine according to Protocol (4) HTS-FT method. 8

9 3-2. Protocol 5: HTS-FT method (Forward Transfection method) The HVJ-E vector suspension (sirna/mirna is incorporated into HVJ-E) that has been prepared with a surface-unprocessed plate can be used for the forward transfection (FT) method (HTS-FT method), in which the suspension is added to the plate seeded with the cells beforehand (cell culture plate). Preparation of HVJ-E suspension (reconstitution of freeze-dried HVJ-E) The tube containing freeze-dried HVJ-E is combined with ice-cooled buffer (0.26 ml). The mixture is gently pipetted, with care taken to avoid bubbling. A homogeneous suspension is thus prepared. The HVJ-E suspension gradually loses activity if the temperature is above 8 C. The suspension should be immediately stored in an ice-cooled bath or in a refrigerator (2-8 C). See page 2 for storage method. Recommended sirna (oligo-type) concentration:1μm(0.05-4μm)(21nt sirna:1μm=1pmol/μl=~0.014μg/μl) Materials to be prepared separately for the HTS protocol: In the case of a shortage in the buffer solution, please use phosphate buffered saline (PBS: free from Ca 2+ and Mg 2+ ). Please use a plate with non-treated surfaces for incubation of suspension cells. Precautions for Use Please use the HVJ-E suspension, the sirna/mirna solution, the Buffer, and the reagents D and E, which have been cooled in ice. Please consistently use a plate with non-treated surfaces for preparation of the HVJ-E vector suspension [Steps (5) to (7)]. Protocol 5: HTS-FT method (in a case of preparation of cells at a cell count corresponding to that in 100 wells of a 96-well plate) Steps (1) through (4) are identical to those of Protocol 4. Step Amount of reagent (1) HVJ-E suspension taken into a micro-test tube HVJ-E suspension:12.5μl (2) Combination with Reagent D and agitation (tapping) Reagent D: 2.5μL (3) Centrifugation at 10,000 g(10,000-12,000rpm) for 5 minutes at 4 C. Supernatant is discarded. (4) Sediment suspended in the buffer (After pipetting 100 μl 10 to 20 times, Buffer:500μL add 400 μl, then pipet once. 11 )]; (5) Add the sirna or mirna solution to each well of the 96-well plate with non-treated surfaces. sirna/mirna solution:5μl (6) Add the suspension prepared in Step [4] to each well (pipetting 1 to 3 suspension prepared in times), and then mix. Step(4):5μL (7) Add the reagent E that has been diluted with the buffer solution by 20-fold to each well, and then mix. 12 (pipetting 1 to 3 times) 1/20 Reagent E: 5μL (8) Add the HVJ-E vector suspension to each well of the plate seeded with HVJ-E vector suspension the cells beforehand, and incubate it under the conditions at 37ºC/5% [(5)+(6)+(7)]: 15μL CO2. 13 (The medium is reviewed hours later, as needed.) Steps (1) through (4) should be performed on ice (To avoid reduction of HVJ-E activity due to temperature rise). The transfection of sirna / mirna into cultured cells is possible even in the presence of serum. When in a state of Step (4), the HVJ-E suspension can be stored under refrigeration (4ºC) for 24 hours. In a case where the procedures (5) to (7) are carried out at room temperature using automatic dispensing equipment, etc., please consistently use each solution that has been cooled in ice, and carry out the procedures in each step promptly to avoid deterioration of the activity of the HVJ-E due to an increase in temperature. The amount of Reagent D added is equal to 1/5 of the HVJ-E suspension [Step (2)]. 11 :If the bottom part of the tube is held, such an action may lead to inactivation of the HVJ-E due to an increase in pellet temperature. Therefore, please pipet the sample by holding the top part of the tube. 12 :Please adequately mix by pipetting, etc., which can affect transfection efficiency. 13 :Please monitor gene silencing efficiency at an appropriate time (e.g., 6 to 72 hours after transfection, although it depends on the experimental system). 9

10 [Amount of reagent for each well plate size] Plate size Forward Transfection (FT) HVJ-E vector suspension [(5)+(6)+(7)] 96-well 15μL 384-well 6μL [Recommended cell density (adherent cells)] Used for transfection under conditions of one-day culture and 50-80% confluency. [Recommended cell density (Suspension cells)] Refer to page 11 for cell density of suspension cells per well. [One-point advice] Because the HVJ-E is adsorbed to a plate with tissue culture treated surface, please consistently use a plate with non-treated surface for preparing the HVJ-E vector suspension. [Troubleshooting] Low efficiency of transfection Efficiency may be increased by the following measures: The concentration of sirna/mirna solution is elevated. The amount of HVJ-E suspension[step (1)] increases. Adequately mix the cells with the HVJ-E vector suspension by pipetting, etc., in Step (8). The use of small-diameter wells such as those of a 384-well plate makes it difficult to thoroughly mix the cells from Step (8) with the HVJ-E vector suspension, leading to reduced transfection efficiency. Ensure that the cells are well-mixed with the HVJ-E vector suspension. If thorough mixing is difficult to achieve, centrifuging at 1500 g for 5 min at 25ºC allows sufficient contact between the cells and the HVJ-E vector, thereby preventing a decrease in transfection efficiency. 10

11 4. sirna/mirna introduction into laboratory animals (in vivo) The protocol shown below pertains to an example in which transfection of mouse organs or tissue (direct injection to organs or tissue) in vivo is attempted. The method of administration, dose level, etc., can vary markedly depending on the species of animals, the type or location of the target organ, and other factors. These conditions may need to be adjusted in individual cases Protocol 6: in vivo Method Preparation of HVJ-E suspension (reconstitution of freeze-dried HVJ-E) The tube containing freeze-dried HVJ-E is combined with ice-cooled buffer (0.26 ml). The mixture is gently pipetted, with care taken to avoid bubbling. A homogeneous suspension is thus prepared. The HVJ-E suspension gradually loses activity if the temperature is above 8 C. The suspension should be immediately stored in an ice-cooled bath or in a refrigerator (2-8 C). See page 2 for storage method. Precautions for Use Please use the HVJ-E suspension, the sirna/mirna solution, and the reagents D and E, which have been cooled in ice. Recommended sirna (oligo-type) concentration: 1μg/μL (21nt sirna:10μm=10pmol/μl= ~0.14μg/μL) Protocol 6: in vivo Method Step Amount of reagent (1) HVJ-E suspension taken into a micro-test tube HVJ-E suspension: 40μL (2) Combination with Reagent D and agitation (tapping) Reagent D: 8μL (3) Centrifugation at 10,000 g(10,000-12,000rpm) for 10 minutes at 4 C. Supernatant is discarded. (4) Sediment suspended in sirna/mirna solution (pipetted times) sirna/mirna solution:10μl (5) HVJ-E vector suspension administered to animals (after dilution with Dose level: Adjusted tailored to physiological saline, etc., as needed) the objective Steps (1) through (4) should be performed on ice (To avoid reduction of HVJ-E activity due to temperature rise). The amount of Reagent D added is equal to 1/5 of the HVJ-E suspension [Step (2)]. [One-point advice] The amount of HVJ-E is approximately 40-80μL for mice and μL for rats. The amount of each reagent used in subsequent steps also needs to be adjusted in proportion to the amount of HVJ-E used. Depending on the features of the experimental system (the site of administration, route of administration, etc.), dose level must be adjusted appropriately by adding buffer, physiological saline, or the like to the HVJ-E vector suspension [Step (5)]. When this product is used in animals, it is as a rule advisable to skip treatment with Reagent C. If the substance introduced needs to be retained in tissue near the site of injection, you may add Reagent C to HVJ-E vector suspension [Step (5)]. Because HVJ-E can be easily adsorbed onto blood cells and be inactivated in vivo, it is advisable to select a route of administration involving less exposure to blood (direct injection to organs or tissue is recommended) or to perform perfusion of the animal prior to administration. 11

12 Precautions for use 1. This product is sold for research purpose only. It may not be used for treatment or other clinical purposes or for intra- and extracorporeal diagnosis in humans or animals. 2. When using this product for recombinant DNA experiments, rules for recombinant DNA experiments (stipulated in relevant statutes in the country of use or set forth by the safety committee of the facility concerned) must be followed, and experiments should only be carried out in laboratories properly equipped with facilities appropriate for recombinant DNA experiments. 3. Experiments using this product must only be carried out by investigators who have been trained in laboratory techniques and have knowledge of and skill in cell culture and genetic engineering. 4. Laboratory staff members working in the area where HVJ-E experiments are occurring should be informed of the properties of HVJ-E, in order to prevent accidents arising from inappropriate handling of it. 5. Although the HVJ (Sendai virus) contained in the HVJ envelope (HVJ-E) of this kit has been inactivated to completely eliminate its proliferative and infective potential, it retains membrane-fusion activity. Therefore, to prevent inhalation, attachment, unintended swallowing, or spread to eyes or nose of the HVJ-E particles, the product must be manipulated within a safety cabinet, wearing appropriate clothing (laboratory overalls) and protective items (plastic or latex gloves, mask, protective eyeglasses, etc.). 6. Do not pipette HVJ-E by mouth. Avoid splashing or generation of aerosols. Avoid contact of skin or mucous membranes with HVJ-E and other kit reagents. In the case of contact with skin or eyes, wash immediately with water. Membrane-fusion activity of HVJ-E is inactivated by autoclaving or treatment with detergent or 70% ethanol. 7. Empty containers of HVJ-E and tools and devices exposed to HVJ-E (pipettes, dishes, chips, etc.) must be handled carefully and disposed of after being autoclaved. 8. Although none of the other reagents contained in the kit is a toxic or powerful substance, they should be handled with protective items (laboratory overalls, gloves, mask, etc.). 9. The HVJ-E suspension has been confirmed by sterility testing to be free of contamination by bacteria or fungi. However, absence of contamination by all microorganisms cannot be guaranteed and appropriate procedures must be followed when using this product. 10. Freeze-dried HVJ-E and the reconstituted suspension should be stored at 2-8 C. Do not use HVJ-E beyond expiration date on label. 11. The proper use of this product is described in the instructions given in this package insert. Manufacturer (Ishihara Sangyo Kaisha, Ltd.) and distributors are not liable for any accident or damage arising from the use of this product which is not in strict compliance with these instructions 12. This product and its use are covered by the claims of one or more patents (including patents pending) and licensed for research use only. It may not be used for any commercial or other purpose or resold after modification or the like without prior written approval from manufacturer (Ishihara Sangyo Kaisha, Ltd.). Handling instructions may be changed. The latest version can be downloaded from the homepage specified below. Manufacturer ISHIHARA SANGYO KAISHA, LTD. 12