QuickTiter™ AAV Quantitation Kit
Contents
1. Part No 314502 One 10 mL bottle QuickTiter Solution C 10X Part No 314503 One 5 mL bottle CyQuant GR Dye 400X Part No 105101 One 50 uL tube QuickTiter AAV DNA Standard Part No 314504 One 500 uL tube containing 100 ug mL AAV DNA Standard Materials Not Supplied Sow ue dq Ze AAV Helper Free System HEK 293 cells and cell culture growth medium Cell culture centrifuge 1X TE 10 mM Tris pH 7 5 1 mM EDTA Fluorescence Plate Reader Storage Store ViraBind AAV Reagent B at room temperature and all other kit components at 4 C until their expiration dates AN CELL BIOLABS INC p D Safety Considerations Remember that you will be working with samples containing infectious virus Follow the recommended NIH guidelines for all materials containing BSL 2 organisms Preparation of Reagents e 1X QuickTiter AAV Wash Solution Prepare a 1X QuickTiter AAV Wash Solution by diluting the provided 5X stock 1 5 in deionized water Store the diluted solution at room temperature e 1X QuickTiter Solution C Prepare a 1X QuickTiter Solution C by diluting the provided 10X stock to 1X with 1X TE Store the diluted solution at room temperature e 1X CyQuant amp GR Dye Estimate the amount of 1X CyQuant GR Dye needed based on the number of assays including AAV DNA standard samples Immediately before use prepare a 1X CyQuant amp GR Dye by diluting the provided 400X stock 1 400 i2. Product Manual QuickTiter AAV Quantitation Kit Catalog Number VPK 145 20 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Viral gene delivery systems include vectors developed from retrovirus RV adenovirus AdV adeno associated virus AAV lentivirus LV and herpes simplex virus HSV AAV belongs to the family of Parvoviridae a group of viruses among the smallest of single stranded and non enveloped DNA viruses There are eight different AAV serotypes reported to date Recombinant AAV 2 is the most common serotype used in gene delivery and it can be produced at high titers with a helper virus or Cell Biolabs AAV Helper Free System AAV 2 can infect both dividing and non dividing cells and can be maintained in the human host cell creating the potential for long term gene transfer Because AAV 2 is a naturally defective virus requiring provision of several factors in trans configuration for productive infection it is considered the safest viral vector to use Recently a new vector AAV DJ was developed using DNA family shuffling to create a hybrid capsid from 8 different AAV serotypes resulting in a vector with significantly higher in vitro infection rates across a variety of cells and tissues Recombinant AAV 2 and AAV DJ vectors can be purified by CsCl gradient ultracentrifugation iodixanol discontinuous gradient ultrac
3. and 1 5 uL of 10X QuickTiter Solution C in a tube 3 Transfer 10 uL of the mixtures including the non heated control sample to a microtiter plate suitable for reading in a fluorometer Add 90 uL of freshly prepared 1X CyQuant GR Dye to each well containing the 10 uL supernatant Read the plate with a fluorescence plate reader using a 480 520 nm filter set 4 Calculate AAV titer based on the standard curve Example of Results The following figures demonstrate typical quantitation results One should use the data below for reference only This data should not be used to interpret actual results 200 20 175 150 15 125 u 100 Td 10 tcc 75 50 5 25 0 0 0 25 50 75 100 125 150 0 2 4 6 8 10 AAV DNA STD ng AAV DNA STD ng Figure 1 AAV 2 DNA Standard Curve The QuickTiter AAV 2 DNA Standard was diluted as described in the Assay Protocol Fluorescence measurement was performed on a SpectraMax Gemini XS Fluorometer Molecular Devices with a 485 538 nm filter set and 530 nm cutoff CELL BIOLABS INC A Calculation of AAV 2 Titer Genome Co GC mL 1 Determine AAV 2 DNA amount 1 Calculate Net RFU Relative Fluorescence Unit Net RFU RFU viral sample RFU negative control corresponding to viral sample 2 Use the standard curve to determine the viral DNA amount of each unknown sample 2 Calculate Viral Titer The average genome size of an AAV 2 is 50
4. emmm 201404610 8 CELL BIOLABS INC JA en 3 Stankowska D L et al 2015 Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma nvest Ophthalmol Vis Sci 56 893 907 4 Li Y et al 2014 Assembly and validation of versatile transcription activator like effector libraries Sci Rep 4 4857 5 Paydar A et al 2014 Extrasynaptic GABAA receptors in mediodorsal thalamic nucleus modulate fear extinction learning Mol Brain 7 39 6 Vinnikov I A et al 2014 Hypothalamic miR 103 protects from hyperphagic obesity in mice J Neurosci 34 10659 10674 7 Ma J et al 2013 RNA interference mediated silencing of Atp6i prevents both periapical bone erosion and inflammation in the mouse model of endodontic disease nfect Immun 81 1021 1030 8 Tao P et al 2013 In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine PNAS 10 1073 pnas 1300867110 License Information This product is provided under an intellectual property license from Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased product and components of the product only in research conducted by the buyer whether the buyer is an academic or for profit entity The sale of this product is expressly conditioned on the buyer not using the product or its components or any materials made using t
5. 00 base therefore 1 ng AAV 2 DNA 1x10 g 5000 base x 330 g base X 6 x 10 3 6 x 10 GC a For unpurified AAV 2 sample Net RFU z RFU viral sample RFU 0 ng mL standard Titer GC mL Dilution Factor X AAV 2 DNA ng X 3 6 x 10 GC ng X 20 uL 10 uL 1 0 mL b For purified AAV 2 sample Net RFU RFU viral sample RFU non heated control sample Titer GC mL Dilution Factor X AAV 2 DNA ng X 3 6 x 10 GC ng X 15 uL 13 5 uL 0 010 mL Calculation Example Purified AAV 2 GFP ViraBind AAV Purification Kit undiluted purified AAV 2 GFP was used as described in Assay Protocol Net RFU 54 6 3 3 51 3 or 28 ng of viral DNA Titer GC mL Dilution Factor X AAV 2 DNA ng X 3 6 x 10 GC ng X 15 uL 13 5 uL 0 010 mL Titer GC mL 1 X 28 ng X 3 6 x 10 GC ng X 15 uL 13 5 uL 1 12 X 10 GC mL 0 010 mL References Rabinowitz J and Samulski R J 1998 Curr Opin Biotechnol 9 470 475 2 Summer ford C and Samulski R J 1999 Nat Med 5 587 588 3 Clark K Liu X McGrath J and Johnson P 1999 Hum Gene Ther 10 1031 1039 Recent Product Citations 1 Orabi A I et al 2015 Dynamic imaging of pancreatic NF B activation in live mice using AAV infusion and bioluminescence J Biol Chem doi 10 1074 jbc M115 647933 2 Oh S M et al 2015 Combined Nurrl and Foxa2 roles in the therapy of Parkinson s disease EMBO Mol Med doi 10 15252
6. 30 minutes for purified AAV Each kit provides sufficient quantities to perform up to 20 tests for unpurified AAV 2 or AAV DJ samples generated from the AAV Helper Free System QuickTiter AAV Quantitation Kit provides an efficient system for rapid quantitation of AAV titer for both viral supernatant and purified virus 2 s CELL BIOLABS INC p Assay Principle How QuickTiter Kit Works 1 Viral Stock Protein RNA DNA AAV 2 2 Nuclei Acid Digestion 3 Virus Capture Bead a AAV 2 4 Denaturation Viral Genome Release and Renaturation Denatured Proteins Viral Nuclei Acid 5 Quantitation Fluorescence 2 Denatured Proteins Viral Nuclei Acid 3 CELL BIOLABS INC Related Products wo oos 2x E XA Ee cgo dq E AAV 100 293AAV Cell Line VPK 140 ViraBind AAV Purification Kit VPK 141 ViraBind AAV Purification Mega Kit AAV 200 ViraDuctin AAV Transduction Kit VPK 109 QuickTiter Adenovirus Titer Immunoassay Kit VPK 110 QuickTiter Adenovirus Titer ELISA Kit VPK 106 QuickTiter Adenovirus Quantitation Kit VPK 112 QuickTiter Lentivirus Quantitation Kit VPK 120 QuickTiter Retrovirus Quantitation Kit Kit Components QS Qv M Se YS SS ViraBind AAV Reagent A Part No 314001 One 0 3 mL tube ViraBind AAV Reagent B Part No 314002 One 1 5 mL tube QuickTiter AAV Capture Matrix Part No 314501 One 1 mL tube QuickTiter AAV Wash Solution 5X
7. ed No part of these works may be reproduced in any form without permissions in writing 9 e CELL BIOLABS INC A
8. entrifugation or Cell Biolabs ViraBind AAV Purification Kit A particular challenge in the delivery of a gene by a viral vector is the accurate measurement of virus titer Traditionally AAV particles are measured by DNA dot blot or similar approaches These methods are time consuming and suffer from a high degree of inter assay variability For highly purified virus samples an optical absorbance at 260 nm has been used to estimate the total number of virus particles However this method cannot be used in an unpurified viral supernatant because other components it contains can contribute to the optical absorbance of 260 nm An ELISA method has been developed by using antibody that only reacts with AAV intact particles however this method measures all AAV particles including the ones lacking genomic DNA Cell Biolabs proprietary QuickTiter AAV Quantitation Kit does not involve cell infection instead it specifically measures the viral nucleic acid content of purified viruses or unpurified viral supernatant sample See Assay Principle The kit is especially useful for determining the supernatant titer before the purification step The kit has a detection sensitivity limit of 1 X 10 GC mL genome copy mL for unpurified AAV 2 or AAV DJ supernatants or 5 X 10 GC mL for purified AAV sample from any serotype which is sufficient for mid or high titer AAV samples The entire procedure takes about 4 hours for unpurified supernatant or about
9. he product or its components in any activity to generate revenue which may include but is not limited to use of the product or its components 1 in manufacturing ii to provide a service information or data in return for payment iii for therapeutic diagnostic or prophylactic purposes or iv for resale regardless of whether they are resold for use in research For information on purchasing a license to this product for purposes other than as described above contact Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 USA or outlicensing lifetech com Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2008 2015 Cell Biolabs Inc All rights reserv
10. hese cells until the monolayer is 70 80 confluent Cotransfect cells with the pAAV RC pHelper and your expression construct according to manufacturer s manual After 48 72 hrs harvest the transfected cells plus culture medium in a conical tube and centrifuge for 5 min at 3000 rpm to pellet the transfected cells Resuspend the cell pellet in 2 5 mL of serum free DMEM Subject the cell suspension to four rounds of freeze thaw cycles by alternating the tubes between the dry ice ethanol bath and the 37 C water bath Collect the AAV supernatant by centrifugation at 10 000 x g for 10 minutes Discard the pellet The viral supernatant can be stored at 80 C or immediately titered or purified Assay Protocol I Unpurified AAV 2 or AAV DJ Samples Note The following procedure is written for quantitation of 1 0 mL of unpurified AAV 2 or AAV DJ supernatant For AAV samples that are less than 1 0 mL add serum free DMEM to the final volume of 1 0 mL Add 10 uL of ViraBind AAV Reagent A to 1 0 mL of viral supernatant mixing well Incubate for 30 minutes at 37 C 3 Incubate ViraBind M AAV Reagent B for 30 60 minutes at 37 C to ensure Reagent B is dissolved Add 50 uL of ViraBind AAV Reagent B to the viral supernatant pre treated with ViraBind AAV Reagent A mixing well Incubate for 30 minutes at 37 C 5 Resuspend the QuickTiter AAV Capture Matrix by inverting and shaking Add 50 uL of matrix to the virus superna
11. n 1X TE For best results the diluted solution should be used within 2 hrs of its preparation Preparation of Standard Curve 1 Create AAV DNA standards from 10 ug mL 5 ug mL 2 5 ug mL 1 25 ug mL 0 ug mL using 1 2 serial dilutions 1X QuickTiter AAV DNA Standard 100 ug mL AAV DNA Solution C Standard Tubes Standard uL uL ug mL 1 10 90 10 2 50 of Tube 1 50 5 3 50 of Tube 2 50 2 5 4 50 of Tube 3 50 1 25 5 50 of Tube 4 50 0 625 6 50 of Tube 5 50 0 313 7 50 of Tube 6 50 0 156 8 50 of Tube 7 50 0 078 9 50 of Tube 8 50 0 039 10 50 of Tube 9 50 0 020 11 50 of Tube 10 50 0 010 12 0 50 0 2 Transfer 10 uL of each dilution including blank to a microtiter plate suitable for reading on a fluorometer Add 90 uL of 1X CyQuant GR Dye to each of the wells containing the 10 uL sample Read the plate with a fluorescence plate reader using a 480 520 nm filter set 5 AN CELL BIOLABS INC Re a Preparation of rAA V Samples The following procedure is suggested for one 15 cm dish or two 10 cm dishes and may be optimized to suit individual needs For best results please refer to your user manual for Cell Biolabs AAV Helper Free System or other system you are using In general each cell produces about 20 000 to 100 000 viral particles under optimized conditions l Use HEK 293 cells that have been passaged 2 3 times prior to transfection Culture t
12. tant Mix the supernatant matrix suspension at room temperature for 30 minutes on a shaker Pellet the AAV Capture Matrix by centrifugation for 10 minutes at 1 000 rpm 8 Carefully remove the supernatant and wash the AAV Capture Matrix with 1 0 mL of 1X QuickTiter AAV Wash Solution Pellet the AAV Capture Matrix by centrifugation for 5 minutes at 1 000 rpm and carefully remove the supernatant Repeat the wash step once and aspirate the final wash To remove the last bit of liquid centrifuge the tube again at 2000 rpm for 30 seconds and remove remaining supernatant with a small bore pipette tip to avoid disturbing the beads 6 JaN CELL BIOLABS INC p 10 Add 20 uL of 1X QuickTiter Solution C and mix with the beads by vortexing for 30 seconds Incubate 1 hr at 75 C Spin down the beads at 12 000 rpm for 30 seconds 11 Transfer 10 uL supernatant to a microtiter plate suitable for fluorometer Add 90 uL of freshly prepared 1X CyQuant GR Dye to well s containing the 10 uL supernatant Read the plate with a fluorescence plate reader using a 480 520 nm filter set 12 Calculate AAV titer based on the standard curve II Purified AAV Sample 1 Mix 13 5 uL of purified AAV sample and 1 5 uL of 10X QuickTiter Solution C in a tube and incubate 1 hr at 75 C Spin briefly to collect condensation Incubate 20 minutes at room temperature 2 Prepare a non heated control sample by mixing 13 5 uL of the same purified AAV sample
Download Pdf Manuals
Related Search