TOOLS sirna and mirna. User guide

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1 TOOLS sirna and mirna User guide

2 Introduction RNA interference (RNAi) is a powerful tool for suppression gene expression by causing the destruction of specific mrna molecules. Small Interfering RNAs (sirnas) are short, double-stranded RNA molecules that bind specific messenger RNA (mrna) molecules and decrease their activities by preventing an mrna from producing a protein. TOOLS sirna oligos undergo vigorous process monitoring and strict quality control, produce under ISO9000 quality standard system. Chemical Modified sirna exhibits greater longevity in cell culture and stability in cell culture and in serum, enhancing the ability for this to be used for in vivo applications and has much more extensional effective time than Standard sirna. TOOLS in vitro/vivo mirna mimics inhibitors are chemically-modified and optimized nucleic acids designed to specifically target the microrna (mirna) molecules in animals. Endogenous micrornas are small, regulatory RNAs that are expressed in animals and plants that affect the translation of target mrnas. The mature nucleotide, single-stranded mirnas specifically target a protein complex to regulate translation at the level of mrna. Specifications Products (1 OD) sirna mirna mimics mirna inhibitor nmol Molecular weight ~13300 ~13300 ~6500 weight 33 ug 33 ug 33 ug Concentration 20 um (20 pmol/ul) 20 um (20 pmol/ul) 20 um (20 pmol/ul) (when adding 125 ul H 2O) (when adding 125 ul H 2O) (when adding 250 ul H 2O) 1

3 Ordering information sirna In vitro sirna or chemically-modified sirna In vivo sirna or chemically-modified sirna Negative Control sirna FAM Negative Control sirna Positive Control sirna sirna set (Guaranteed >70% knock-down in mrna level) 3 x 1OD target sirna 1 x 0.5OD negative control sirna 1 x 0.5OD FAM labeled negative control sirna 1 x 0.5OD positive control sirna Chemically-modified sirna set(guaranteed >70% knock-down in mrna level) 3 x 1OD Chemically-modified target sirna 1 x 0.5OD negative control sirna 1 x 0.5OD FAM labeled negative control sirna 1 x 0.5OD positive control sirna 2OD/4OD/8OD request 1OD 1OD 1OD mirna mimics & inhibitors In vitro mirna mimics In vitro mirna mimics negative control In vitro chemically-modified mirna inhibitor In vitro chemically-modified mirna inhibitor negative control In vivo chemically-modified mirna mimics In vivo chemically-modified mirna inhibitor In vivo mirna mimics & inhibitor negative control 2OD/4OD 2OD 2OD/4OD 2OD request request request 2

4 sirna transfection guide: Cell Culture Before Transfection It is advised that before starting your transfection experiment, put your cells on your cell plate, then add proper culture medium, lastly incubate cells for 24 hrs to be 70%-90% by confluence. Transfection reagent: TOOLSoothFect transfection reagent (Cat.No. NFT-KA00) Culture vessel Surface Area (mm 2 /well) Cell Density Volume of plating Medium(ul/well) 96-well x x ul 48-well x x ul 24-well x x ul 12-well x x ml 6-well x x ml 35 mm x x ml 60 mm x x ml Culture vessel sirna Volume of plating Transfection reagent mediun (ul/well) 96-well 5 pmol 100ul 0.25 ul 24-well 20 pmol 500ul 1 ul 12-well 40 pmol 1ml 2 ul 6-well 100 pmol 2ml 5 ul 35 mm 100 pmol 2ml 5 ul 60 mm 600 pmol 5ml 10 ul mirna mimics or inhibitor transfection guide: mirna Culture Dish Transfection reagent (mimics or inhibitor) Cell density (cells/well) Final volume (per well) 96-well ul 3 pmol ml 24-well ul 15 pmol ml 12-well 1-3 ul 30 pmol ml 6-well 2-5 ul 75 pmol ml 3

5 In vivo sirna delivery guide: Delivery route (dissolve in vivo sirna in PBS or RNase free water or OptiMEM) Intravenous Intraperitoneal Intracranial Systemic delivery dosage: nmol/g body weight Mice (body weight 15-20g): 3-4 nmol (40-50ug) Rat (body weight g): nmol ( ug) A sirna dose of 5mg/kg/day is recommended as a starting point for experiments. In vivo mirna (mimics or inhibitor) delivery guide: Delivery route (dissolve in vivo mirna in PBS or normal saline:) Intravenous Intraperitoneal Intracranial 5-80ug/g body weight or 200nmol(3 times) Local delivery: 1-10nmol(0.5-4OD), volume: 30ul~100ul; TOOLS mirna modification example: hsa-mir-137 agomirs sense : 5' UUAUUGCUUAAGAAUACGCGUAG 3' antisense: 5' AsCsGCGUAUUCUUAAGCAAUAsAsUsUs-Chol- 3' Storage Store TOOLS sirna or mirna at 20 C 4

6 sirna or mirna FAQs : What is the best method of delivering sirnas into the cell? There are several methods that are used for delivering sirnas into cells including lipid-based transfection, electroporation, calcium phosphate co-precipitation, microinjection and vector delivery techniques. The choice between these methods is often a result of several factors including the ability of the cells to tolerate the delivery method, susceptibility to viral infection, and the growth properties of the cells. Although lipid-based transfection is one of the more commonly used methods for adherent cells, suspension cells are often more difficult to transfect and generally have higher rates of delivery with electroporation techniques. Is there a quick method for monitoring transfection efficiency? TOOLS has developed several options including a family of fluorescent-labeled sirnas and a novel RNA-based reagent known as sirna transfection Control. The uptake of sirnas can be visualized with the appropriate filters on a confocal microscope or by flow cytometry. Alternatively, fluorescent-labeled sirna is cytotoxic when successfully delivered into cells. Cells that have efficiently taken up this transfection control typically undergo apoptosis within 24 to 48 hours. This phenotypic outcome can easily be monitored using standard cell viability methods (e.g., alamarblue, MTT cytotoxicity, Trypan Blue dye exclusion, JC1 dye, or other appropriate assays). What is the best time-frame for monitoring sirna-dependent decreases in target mrna expression levels? In target protein levels? Generally, target mrna levels are decreased after 24 hours post-transfection following transfection of a gene-specific sirna duplex. However, maximal silencing may be reached at a later time point, so it is advisable to assay target mrna in a time-course study. In most cases, silencing will be maximal at 24 to 48 hours following transfection. Cellular target protein levels should be examined starting at 24 hours and assayed until a minimum level is noted, often 48 to 96 hours or greater. As always, it is important to verify transfection efficiency using an appropriate positive control. If there is no decrease in protein levels within this time frame, it may be necessary to perform a second sirna transfection, use a stabilized sirna, or develop a vector-based silencing cassette that can continuously produce the sirna for extended periods of time. 5

7 TOOLS shrna Vector Pre-cloned and ready-made shrna expression plasmids for long-term gene silencing

8 Introduction RNA interference (RNAi) is a powerful tool for suppression gene expression by causing the destruction of specific mrna molecules. Small Interfering RNAs (sirnas) are short, double-stranded RNA molecules that bind specific messenger RNA (mrna) molecules and decrease their activity, for example by preventing an mrna from producing a protein. A small hairpin RNA or short hairpin RNA (shrna) is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNAi. TOOLS provides the most advanced and convenient shrna expression plasmid that express functional sirna when transfected into mammalian cells. TOOLS shrna vector is a highly efficient ready-to-use vector that achieve lasting inhibition of target gene expression. Users only need to provide gene name or ID and select one of our 10 vectors. We produce ready-to-use TOOLS shrna Vector with desired sirna insert for our customer. Ordering information Ready-to-use TOOLS shrna Vector (desired sirna insert has been cloned) Product Promoter Selection marker Size pgpu6 U6 50µg pgph1 H1 50µg pgpu6/neo U6 Neo 50µg pgph1/neo H1 Neo 50µg pgpu6/hygro U6 Hygro 50µg pgph1/hygro H1 Hygro 50µg pgpu6/gfp/neo U6 Neo 50µg pgph1/gfp/neo H1 Neo 50µg pgpu6/rfp/neo U6 Neo 50µg pgph1/rfp/neo H1 Neo 50µg Neo: Neomycin resistance gene. Hygro: Hygromycin B resistance gene. TOOLS shrna Vector set Each packset contains 4 shrna vectors designed for a target gene Guarantee: one of shrna vectors shows 70% inhibition efficiency in mrna level Set composition Number shrna vector for a target gene 4 positive control shrna vector 1 negative control shrna vector 1 Each plasmid contains 50µg Storage Store TOOLS shrna vector at 20 C 1

9 Transfecting TOOLS shrna vector into Mammalian Cells We recommend using TOOLSoothFect transfection reagent (Cat.No. NFT-KA00) to deliver TOOLS shrna vectors into mammalian cells with high efficiency and minimal toxicity. It is suitable for the transfection of a wide variety of cell types. Follow the instructions for using TOOLSmartFect Transfection Reagent provided with the product or refer to the protocol below: 1. For each well, add 0.5 ml of normal growth medium (antibiotic does not influence the result) freshly 2 hours before transfection. 2. For each well, dilute 0.5 μg of DNA in 50 μl of DMEM without serum in a tube, and mix gently. 3. Add 1 μl of TOOLSoothFect into another tube with 50 μl of DMEM without serum, and mix gently. 4. Add TOOLSoothFect /DMEM into DNA/DMEM solution. Mix by vortexing for 5-10 seconds. 5. Incubate for ~15 minutes at room temperature to allow for TOOLSoothFect /DNA complexes self-assembly. 6. Add the 100 μl TOOLSoothFect /DNA mix drop-wise to the cells in each well and homogenize by gently swirling the plate. 7. Return the plates to the cell culture incubator. 8. Check transfection efficiency 24 to 48 hours post transfection. Culture Dish Surface Area (cm 2 ) Cell Number Medium Volume (ml) Plasmid (μg) TOOLSoothFect (μl) Diluent Volume (μl) 96-well x well x well x well 4 1-2x Well/35 mm x mm/t x mm/t x mm/t x

10 Using the Positive and Negative Controls The Negative Control plasmid supplied with the set is a circular plasmid encoding a hairpin sirna whose sequence is not found in the mouse, human, or rat genome databases. It is ready-to-transfect and can be used to control for the effects of introducing the TOOLS shrna vector into cells. Cells transfected with the TOOLS shrna vector expressing your target-specific sirna should be compared to cells transfected with the corresponding Negative Control. For any RNAi experiment, it is important to include a culture that is transfected with a negative control plasmid as a basis for analysis of gene knockdown. The optimal negative control insert for expression analysis in a gene silencing experiment is the scrambled sequence of your gene specific sirna. The product of the positive control vector contains a sirna template targeting GAPDH. This construct can be used to optimize the transfection procedure. Use GAPDH and the Negative Control to transfect cells, and monitor cell viability and gene silencing of GAPDH to identify optimal transfection conditions. When successfully transfected and expressed, the GAPDH sirna reduces both the mrna and protein levels of GAPDH in human cell lines. This slows the growth rate of the cells and reduces the rate of cell proliferation of most cell types. To assess whether sirna-mediated gene silencing is occurring, levels of GAPDH RNA, levels of GAPDH protein, and/or cell proliferation can be monitored. Any of the following assays for assessing sirna-mediated reduction in GAPDH gene expression can be used: a. Quantitate mrna levels by Northern analysis or real-time PCR. GAPDH mrna levels are typically reduced 50 90% 48 hr after transfection. b. Analyze protein levels by Western blot, immunohistochemistry, or immunofluorescence. GAPDH protein levels are typically reduced 50 90% 48 hr after transfection. c. Look for a reduction in cell proliferation caused by GAPDH knock down. Although it is less direct than looking at GAPDH mrna or protein levels, a reduction in GAPDH activity can be assessed by measuring cell proliferation. Depending on cell type, there should be a >40% reduction in cell number hours after transfection. 3

11 TOOLS shrna vector Maps 4

12 5

13 TOOLSilent shrna Vector Product Name: TOOLSilent shrna 50 ug Cat. Number: TOOLSILSHRNA Key features: Dual-function of TOOLSilent shrna vector (Effective transfection of shrna vector and transduction of lentiviral vector) Packaged lentiviral shrna vector exerts higher ability to integrate into the genome of primary cells, stem cells and non-dividing cells. Customized lentiviral packaging service (in vivo and in vitro) Insert size: ~4kb Low cytotoxicity

14 TOOLSilent Lentiviral Expression System TOOLSilent Lentiviral Expression System consists of: (1) Lentiviral expression vector (TOOLSilent shrna Vector) (2) Lentiviral packaging plasmids (3) Pseudoviral particle producer cell The expression vector comprises required lentiviral packaging, transfection, stable integration of the genetic information. Lentivirus packaging plasmid provides all necessary proteins for transcription and encases an RNA copy of the expression construct into recombinant pseudoviral particles. The pseudoviral particles are secreted by producer cells in culture media and can be directly transduced into target cells.

15 TOOLSilent lentivirus packaging service (in vivo) For in vivo experiments, TOOLS provides high-quality ready to use lentivirus. Optional A set of four shrnas (three target shrna plus one negative control shrna) lentiviruses are designed based on customer s needs (gene name or ID) using high efficient software. The recommended concentration of TOOLSilent lentivirus is TU/ml ( ul mouse tail vein injection) We synthesize DNA double strand sequences and linear process the vectors, link the shrna sequence with the vector, and screen the target recombined target, sequence the vector, and provide the recombined lentivirus shrna vectors to our customers.

16 TOOLSilent Lentivirus Packaging Service (in vitro) TOOLSilent Lentivirus can efficiently introduce and stably express lentiviral constructs in primary cells, stem cells, non-dividing cells and cells are difficult to transduce. A set of four shrnas (three target shrna plus one negative control shrna) lentiviruses Based on the gene information (sequence or Gene ID) provided by the users, we designed the shrna sequences and package with highly efficient, transduction-ready lentiviruses. Optional A set of four shrnas (three target shrna plus one negative control shrna) lentiviruses Procedures (1) Target sequences screening and shrna oligonucleotides designing (2) insert designed oligonucleotides into lentiviral vectors (3) using packaging cells to produce shrna lentivirus (4) transducing target cells with shrna lentivirus Users can select one of the following vectors: PGLV1/U6/GFP PGLV2/U6/Puro

17 PGLV3/H1/GFP+Puro PGLV9/U6/RFP PGLV10/U6/RFP/Puro

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