USER GUIDE
KDalert™ GAPDH Assay Kit
Catalog Numbers AM1639 and AM1639M
Publication Number 1639MRevision D
For Research Use Only. Not for use in diagnostic procedures.Theinformation in this guide is subject to change without notice.
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Contents
3KDalert™ GAPDH Assay Kit User Guide
■ KDalert™ GAPDH Assay Kit . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 5
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 5Product description . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 5Procedure overview . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 7
Reagents provided with the kit and storage conditions . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Materials not provided with the kit . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 9
KDalert™ GAPDH Assay procedure . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 10Before you start . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 10KDalert™ standard fluorescence procedure .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 12Alternate procedures . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 13
Data interpretation . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 15Determining optimal transfectionconditions using fluorescence data . . . . . . . . . . . . . . . .. . . 15Determining optimal transfection conditions usingcolorimetric data . . . . . . . . . . . . . . . . . . . 18
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 19General troubleshooting . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 19Fluorescence assay . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 19
■ APPENDIX A Supplemental information . . . . . . . . . . . . .. . . . . . . . . . . . . 21
Related products available from Life Technologies . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Quality control . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 21Functional testing . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 21
■ APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 23
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 23
Biological hazard safety . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 24
Documentation and support . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 25
Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 25
Obtaining support . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 25
Limited product warranty . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 25
4 KDalert™ GAPDH Assay Kit User Guide
Contents
5KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit
IMPORTANT! Before using this product, read and understand theinformation in the “Safety” appendix in this document.
Introduction
Product description
The KDalert™ GAPDH Assay Kit is a rapid, convenient,fluorescence-based method for measuring the enzymatic activity ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured cellsderived from human, mouse, and rat. The KDalert™ GAPDH Assay Kit isdesigned to facilitate identification of optimal siRNA deliveryconditions by assessment of GAPDH expression and knockdown at theprotein level.
GAPDH: the perfect target for siRNA transfection optimizationexperiments
The GAPDH gene is generally recognized as an ideal target forsiRNA transfection optimization experiments because it is a“housekeeping” gene that is ubiquitously expressed at relativelyconstant levels. This characteristic provides a two-fold utilityfor siRNA transfection experiments.
First, GAPDH gene expression can be readily knocked down in manydifferent cell types by delivery of a validated control GAPDH siRNA(for example, Silencer® Select GAPDH Control siRNA). Thus, theefficiency of siRNA delivery can be monitored by measuring thereduction in GAPDH mRNA or protein levels in cells transfected withGAPDH siRNA relative to cells transfected with a negative controlsiRNA (for example, Silencer® Select Negative Control #1, Cat. nos.4390843 and 4390844).
Second, GAPDH expression serves as a marker for identifyingcellular toxicity resulting from transfection. Transfectionconditions that are least toxic to a given cell line can beidentified by transfecting cells with a negative control siRNA andevaluating whether the transfection results in a decrease inendogenous GAPDH mRNA or protein levels.
The KDalert™ advantage
Current methodology for monitoring gene expression and knockdownat the mRNA level, for example, through qRT-PCR, requires RNAisolation, is time-consuming, and is relatively expensive. TheKDalert™ GAPDH Assay provides a rapid and inexpensive assessment ofGAPDH expression through measurement of GAPDH enzyme activity incell lysates. Thus, the KDalert™ kit provides an ideal tool formonitoring transfections that use GAPDH control siRNA.
6 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit Introduction
How the assay works
GAPDH is a tetrameric enzyme, composed of 36 kD proteinsubunits. It catalyzes the oxidative phosphorylation ofglyceraldehyde-3-phosphate (G-3-P) to bisphosphoglycerate(BPG):
The KDalert™ GAPDH Assay measures the conversion of NAD+ to NADHby GAPDH in the presence of phosphate and G-3-P. Under therecommended assay conditions, the rate of NADH production isproportional to the amount of GAPDH enzyme present. Thus the assaycan be used to accurately determine the amount of GAPDH protein ina sample.
Experimental parameters
The standard KDalert™ GAPDH Assay can be used to accuratelyquantitate GAPDH activity in extracts derived from 2,000–12,000cells. Modifications of the procedure allow detection in as few as500 cells, or with extracts prepared from >12,000 cells. Theassay can be used with either adherent or nonadherent cells in avariety of plate formats.
Interpreting the data
The KDalert™ GAPDH Assay Kit measures GAPDH siRNA-induced geneknockdown at the protein level. In general, successful knockdownwill cause a more dramatic reduction in target mRNA levels than intarget protein levels (figure below), probably because proteinknockdown is influenced by the rates of target protein synthesisand turnover, in addition to the rates of target mRNA synthesis andturnover. For housekeeping genes such as GAPDH, the rate of celldivision and concomitant synthesis of the housekeeping proteinduring the transfection experiment also have an impact on knockdownlevels.
The following figure also shows that the relative effects oftransfection parameters on GAPDH enzyme activity parallel thosemeasured by qRT-PCR. Thus, the KDalert™ GAPDH Assay can be used toidentify optimal transfection conditions.
G-3-P + PO4-2 + NAD+ BPG + NADH + H+GAPDH
7KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay KitIntroduction
Figure1 Comparison of GAPDH enzyme activity and qRT-PCRdata. The indicated number of cells from 3 different cell lineswere transfected with Silencer® Select GAPDH siRNA and Silencer®Select Negative Control #1 siRNA. The remaining GAPDH expression 48hr after transfection for GAPDH siRNA-transfected cultures wascalculated as a percentage of expression of Negative ControlsiRNA-transfected cultures. The KDalert™ GAPDH Assay was used tomeasure GAPDH enzyme activity, and qRT-PCR was used to measureGAPDH mRNA.
This protocol provides guidance for using the obtained data toidentify optimal transfection conditions. Optimized transfectionconditions provide the highest levels of GAPDH knockdown whileminimizing toxicity associated with chemical transfection agents orelectroporation.
Procedure overview
The KDalert™ GAPDH Assay is straightforward and requires about10 minutes of hands-on time. Although it can be used as a generaltool to measure GAPDH enzyme activity, its primary application isfor measurement of GAPDH siRNA-induced knockdown and thusassessment of siRNA delivery. For this application, cultured cellsthat have been previously transfected with GAPDH or negativecontrol siRNA are lysed in the culture dish using a one-steplysis/assay buffer. An assay Master Mix is added to an aliquot ofthe lysed cells, and the fluorescence of the samples is measuredusing a fluorescence plate reader. Modifications of the procedureallow colorimetric, rather than fluorescence, measurement of GAPDHactivity.
For detailed procedures, see sections “KDalert™ standardfluorescence procedure” on page 12 and “Alternate procedures” onpage 13.
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8 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit Introduction
Figure2 KDalert™ GADPH Assay procedure overview.
1. Remove culture medium from transfected cells
2. Add 200 µL KDalert™ Lysis Buffer to each sample well
3. Incubate at 4°C for 20 minutes, then pipet the cell lysate upand down 4–5 times
4. Transfer 10 µL of sample to a 96-well plate
5. Add 90 µL of KDalert™ Master Mix to each sample
6. Measure the increase in fluorescence at room temperature
Cell Lysis
GADPH Enzyme Assay
Data Collection
9KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay KitReagents provided with the kit andstorage conditions
Reagents provided with the kit and storage conditions
Reagents for at least 375 fluorescence KDalert™ GAPDH Assays aresupplied.
Materials not provided with the kit
See AlsoAvidin-Biotin Interaction - UKDetermination of seroprevalence and kinetics of humoral response using mpox virus A29 protein• 96-well assay plates. We recommend black microplates (forexample, Fisher Scientific 1256630) for the fluorescence-basedassay. Clear polystyrene plates such as for culturing cells (werecommend BD Falcon™ #353072 or Greiner Bio-One CellStar® #655180)can also be used for the fluorescence assay and are required forthe alternate colorimetric assay.
• GAPDH siRNA.We recommend Silencer® Select GAPDH siRNA, Cat.nos. 4390849 (5 nmol) and 4390850 (40 nmol) (human, mouse, andrat)
• Negative control siRNA.We recommend Silencer® Select NegativeControl siRNAs, Cat. nos. 4390843 or 4390846 (5 nmol) and 4390844or 4390847 (40 nmol)
• Fluorescence plate reader (suggested) or UV/vis plate reader.•General lab equipment and supplies such as appropriate tubes,pipettors (8- or
12-channel pipettor suggested), vortex mixer, etc.• Tissueculture supplies and equipment including the human, mouse, or ratcells
under study, transfection agents, culture medium, and cultureplates and equipment.
Amount Component Storage
110 mL KDalert™ Lysis Buffer†
† KDalert™ Lysis Buffer is available separately as Cat. no.AM8790G.
–20°C or 4°C
38 mL Solution A –20°C
300 µL Solution B –20°C
200 µL Solution C –20°C
100 µL GAPDH Enzyme Below –70°C
10 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit KDalert™ GAPDH Assay procedure
KDalert™ GAPDH Assay procedure
Before you start Procedure notes• The KDalert™ GAPDH Assay Kitprovides an assessment tool for transfection
optimization experiments using Silencer® Select GAPDH ControlsiRNA. You will also need to develop an experimental strategy fortransfection optimization. Variables such as choice of transfectionagent, transfection agent volume, cell number, and inclusion and/ortiming of a post-transfection media change can be tested andcontrolled to identify conditions that maximize uptake ofintroduced nucleic acid and minimize cellular toxicity.
• The KDalert™ GAPDH Assay Kit is designed to measure genesilencing or knockdown of GAPDH, at the protein level, insiRNA-transfected cultures 48 hours after transfection. We havefound that a 48-hour wait after transfection provides a goodbalance between reliable detection of protein knockdown and theelapsed time required to see such knockdown. Slightly higher levelsof protein knockdown are typically seen 3 days aftertransfection.
• We include GAPDH Enzyme for construction of a standard curve(see Figure 3 on page 15). Including a standard curve in yourexperiments assures that the GAPDH activity measured in the samplesfalls in the 100-fold linear range of the assay. However, byfollowing the protocol exactly, the KDalert™ GAPDH Assay can beused to compare transfection optimization conditions reliablywithout including a standard curve.
Two days before the assay: transfect mammalian cells with GAPDHor Negative Control siRNA
Transfect separate wells of a 96-well culture plate containing2,000–12,000 human, mouse, or rat cells per well with GAPDH orNegative Control siRNA. See “Alternate procedures” on page 13 ifyour experiment requires other plating formats or if you areconducting the colorimetric assay.
Prepare KDalert™ Master Mix
1. On the day of the assay, 2–3 days after transfection,assemble a KDalert™ Master Mix as described in the following table.Prepare Master Mix only for the samples to be assayed that day plus5–10% overage (90 µL KDalert™ Master Mix per reaction).
2. Mix thoroughly by inversion or gentle vortexing.The KDalert™Master Mix can be stored at room temp for
11KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay KitKDalert™ GAPDH Assay procedure
(Optional) Prepare serial dilutions of GAPDH Enzyme
To prepare a standard curve, make serial dilutions of the GAPDHEnzyme in KDalert™ Lysis Buffer over a 100-fold concentrationrange.
1. Thaw GAPDH Enzyme at room temperature, then immediately placeon ice.
2. Mix 4 µL of GAPDH Enzyme with 396 µL KDalert™ Lysis Buffer tocreate a GAPDH Working Stock (see the following table), then use itto prepare serial dilutions of GAPDH as shown in the followingtable. Keep diluted GAPDH on ice until used.
Adjust the fluorescence plate reader settings
Turn on the fluorescence plate reader and set the dataacquisition parameters as follows:
• Set the excitation wavelength at 560 nm and the emissionwavelength at 590 nm.If the fluorometer uses preset filters, usethe filters closest to these wavelengths. We have found thatexcitation and emission filters set to 545 and 575 nm,respectively, work well for the assay.
• Set the plate reader to kinetic mode, if available. • Set thegain to autoscale, if available. Alternatively, set the gain tomedium
initially. • Set the temperature to room temperature.
KDalert™ Lysis Buffer GAPDH Enzyme or dilution Dilution
GAPDH concentration
396 µL 4 µL GAPDH Enzyme GAPDH Working Stock
0.26 U/mL
50 µL 50 µL GAPDH Working Stock
1:2 0.133 U/mL
50 µL 50 µL 1:2 dilution 1:4 0.067 U/mL
50 µL 50 µL 1:4 dilution 1:8 0.033 U/mL
50 µL 50 µL 1:8 dilution 1:16 0.017 U/mL
50 µL 50 µL 1:16 dilution 1:32 0.0083 U/mL
50 µL 50 µL 1:32 dilution 1:64 0.0042 U/mL
50 µL 50 µL 1:64 dilution 1:128 0.0021 U/mL
12 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit KDalert™ GAPDH Assay procedure
KDalert™ standard fluorescence procedure
Use this procedure with the following experimentalparameters:
• 2,000–12,000 human, mouse, or rat cells plated per well of96-well plate(s)• Detection using a fluorescence plate reader
Alternate procedures for different amounts of cells, and forreading the assay colorimetrically, are provided in the nextsection.
1. Remove culture medium from transfected cells48 hours aftersiRNA transfection, aspirate the culture medium from transfectedcells.
STOPPING POINT. The culture plate can be stored at –80°C afterremoving the culture medium. Thaw frozen cells on ice beforeproceeding.
2. Add 200 µL KDalert™ Lysis Buffer to each sample wellAdd 200µL KDalert™ Lysis Buffer to each sample well following the volumeguidelines in the table below.
3. Incubate at 4°C for 20 minutes, then pipet the cell lysate upand down 4–5 times
a. Incubate at 4°C for 20 minutes to lyse thecells.Alternatively, the lysis can be incubated on ice instead ofat 4°C.
b. Pipet the cell lysate up and down 4–5 times (or shake theplate for 20 seconds at room temp) to hom*ogenize the lysate.
4. Transfer 10 µL of sample to a 96-well plateTransfer 10 µL ofeach lysate or GAPDH Enzyme dilution (including the GAPDH WorkingStock) to the wells of a clean 96-well plate. 96-well platerecommendations are provided in section “Materials not providedwith the kit” on page 9.We recommend testing the GAPDH Enzymedilutions in duplicate.
5. Add 90 µL of KDalert™ Master Mix to each sampleWorkingquickly, add 90 µL of KDalert™ Master Mix to each sample. It isimportant that all samples receive the KDalert™ Master Mix atapproximately the same time. We recommend using a multichannelpipettor to dispense the KDalert™ Master Mix quickly.
13KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay KitKDalert™ GAPDH Assay procedure
6. Measure the increase in fluorescence at roomtemperatureMeasure the increase in fluorescence of the samples atroom temperature.The fluorescence plate reader settings areprovided in section “Adjust the fluorescence plate reader settings”on page 11.If the plate reader is capable of real-time kineticmeasurements, immediately measure the increase in fluorescence overa 4 minute interval, collecting data every 1–2 minutes.For endpointfluorescence measurements, measure the fluorescence immediatelyafter adding the KDalert™ Master Mix, then allow the reaction toproceed for 4 minutes at room temperature and measure thefluorescence again. Subtract the initial fluorescence reading fromthe second reading to determine the fluorescence increase for eachsample.Samples with relatively high levels of GAPDH activity willacquire a magenta color approximately 15 minutes after the KDalert™Master Mix is added to the samples. This color does not affect thefluorescence reading and can be considered a visible indicator ofGAPDH activity.
Alternate procedures
For 500–2,000 cells plated per well
For 500–2,000 cells plated per well, follow the standardprocedure detailed in section “KDalert™ standard fluorescenceprocedure” on page 12 with the following modifications:
• In step 2., use 100 µL of KDalert™ Lysis Buffer.• In step 4.,transfer 20 µL of cell extract to a clean 96-well microplate.• Instep 5., add 80 µL Master Mix to each well.
For more than 12,000 cells plated per well
For more than 12,000 cells plated per well, follow the standardprocedure detailed in section “KDalert™ standard fluorescenceprocedure” on page 12 with the following modifications according tothe type of plates used for culturing the cells.
• 96-well platesIn step 4., transfer 2.5 µL of each cell extractto a clean 96-well microplate containing an additional 7.5 µLKDalert™ Lysis Buffer per well.
• 24, 12, or 6-well plates or culture dishesIn step 2., use theKDalert™ Lysis Buffer volumes shown in the following table (thismay require more KDalert™ Lysis Buffer than is supplied with thekit; Cat. no. AM8790G).
Plate format KDalert™ Lysis Buffer
24-well 1 mL per well
12-well 2 mL per well
6-well 4 mL per well
14 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit KDalert™ GAPDH Assay procedure
Colorimetric assay procedure• The colorimetric assay is slightlyless sensitive than the fluorescence assay, so the
reaction volume is increased from 100 µL to 200 µL to improvethe signal. • A water + Master Mix control is used as the referencefor determining GAPDH
activity. • Clear 96-well assay plates are required.• Absorbanceat 615 nm is measured using a UV/visible plate reader.
1. Follow the pre-assay steps described in section “Before youstart” on page 10, except:
• Plate 4,000–12,000 cells per well in 96-well microplates foryour siRNA transfections
• Set up the visible plate reader to read the absorbance of eachsample at 615 nm
• Prepare enough Master Mix to use 180 µL per reaction
2. 48 hours after transfection, remove the culture medium fromthe cells.The plate can be stored at –80°C. Thaw frozen cells onice before proceeding.
3. Add 100 µL KDalert™ Lysis Buffer to each sample.
4. Incubate on ice or at 4°C for 20 minutes.
5. Pipet the cell lysate up and down 4–5 times (or shake platefor 20 seconds at room temperature) to hom*ogenize the lysate.
6. Transfer 20 µL aliquots of each cell extract, GAPDH Enzymedilution (including Working Stock), or water (water + Master Mixcontrol) to wells in a clean 96-well microplate. We recommend usingduplicate aliquots of each GAPDH Enzyme dilution or water + MasterMix control. Use BD Falcon™ #353072 or Greiner Bio-One CellStar®#655180 clear polystyrene plates.
7. Add 180 µL of Master Mix, and allow the reaction to proceedat room temperature for 15 minutes.
8. Measure the absorbance of each reaction at 615 nm. Use awater reference blank in the measurement.GAPDH activity isindicated by a reduction in A615.Some samples may acquire a magentacolor over 15 minutes.
15KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay KitData interpretation
Data interpretation
Determining optimal transfection conditions using fluorescencedata
Calculating GAPDH activity using fluorescence data
For the KDalert™ GAPDH Assay, the GAPDH activity in each sampleis defined as the fluorescence increase over 4 minutes,Δfluorescence (in arbitrary units, calculated automatically with aplate reader set to kinetic mode, or manually by subtracting thefluorescence reading at t0 from that at t4 min).
Note that the magenta color acquired by some samples will notaffect the fluorescence reading.
Standard curve for GAPDH Enzyme
The following figure shows a typical standard curve obtained fora GAPDH Enzyme dilution series. The observed GAPDH activity shouldshow a linear relationship to the amount of enzyme present over a100-fold range.
Figure3 Typical standard curve for GAPDH enzyme. GAPDHEnzyme standard was diluted as described in the table on page 11assayed in duplicate according to the Standard FluorescenceProcedure using a fluorescence plate reader in kinetic mode.
Calculating % remaining gene expression and % knockdown
The percent remaining gene expression for a given transfectioncondition can be determined from the ratio of the fluorescenceincrease for samples transfected with GAPDH siRNA to thefluorescence increase for samples transfected with negative controlsiRNA:
where ΔfluorescenceGAPDH and ΔfluorescenceNeg are the meanfluorescence increases per unit time (for a given transfectioncondition) for samples transfected with GAPDH siRNA and negativecontrol siRNA, respectively (Figure 4A on page 17).
R = 0.9997
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0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
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% remaining expression 100 x ΔfluorescenceGAPDH
ΔfluorescenceNeg--------------------------------------------=
16 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit Data interpretation
The level of gene knockdown for a given transfection conditionis calculated from the percent remaining gene expression (Figure4B):
or
Transfection-associated toxicity
Many transfection agents are somewhat toxic to cells. SinceGAPDH enzyme levels per cell are fairly constant in untransfectedcells or in cells transfected with negative control siRNA, GAPDHactivity in negative control siRNA-transfected cells(ΔfluorescenceNeg) can be used as an indicator of cell number, orof relative viability, under the transfection conditions beingtested.
Specifically, in samples transfected with negative controlsiRNA, the higher the ΔfluorescenceNeg, the less toxic thetransfection conditions (Figure 4C).
Optimal transfection conditions
Typically, there is an inverse relationship between transfectionefficiency (and associated gene knockdown) and cell viability,because many transfection agents are somewhat toxic to cells.Optimal transfection conditions are those which maximize specificgene knockdown while minimizing transfection-associatedtoxicity.
In robust cell lines, for example, HeLa, high levels of targetknockdown can be achieved while maintaining high cell viability. Inless robust lines, a more realistic goal is to strike an acceptablebalance between the relative cell toxicity and the level ofknockdown that can be achieved.
A useful way to describe the balance of transfection efficiencyand cell toxicity is the term Optimal Balance Factor, or OBF. TheOBF is calculated for each transfection condition as follows:
Typically, optimal transfection condition(s) are those whichexhibit the highest OBF value (Figure 4D)
For example, in the optimization experiment shown in Figure 4,maximum GAPDH knockdown (minimum remaining expression) was seenusing 0.6 µL transfection agent and plating 4,000 cells per well(panel B). However, relative viability was greatest at 0.3 µLtransfection agent (panel C). The OBF calculated for each conditionindicates that 0.3 µL of transfection agent and 4,000 cells perwell provides the best overall balance between target knockdown andcell viability (panel D).
% knockdown 100 % remaining expression–=
% knockdown 100 100ΔfluorescenceGAPDH
ΔfluorescenceNeg--------------------------------------------×⎝⎠
⎛ ⎞–=
OBF ΔfluorescenceNeg x % knockdown=
ΔfluorescenceNeg x 100 % remaining expression–[ ]=
17KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay KitData interpretation
Figure4 Optimizing transfection conditions using theKDalert™ assay. UMR106 cells were reverse-transfected with eitherGAPDH or Negative Control #1 siRNA using the indicated amount ofsiPORT™ NeoFX™ Transfection Agent and plating at 4,000 or 8,000cells per well. GAPDH activity was measured 48 hr aftertransfection using the KDalert™ GAPDH Assay. Panel A: GAPDHactivity in arbitrary fluorescence units. GAPDH activity is notnormalized for cell number, so higher activity is seen in allsamples plated at 8,000 cells/well.Panel B: Remaining GAPDHactivity in GAPDH siRNA-transfected cultures, relative to NegativeControl #1 transfections. Arrow indicates transfection conditionresulting in lowest remaining expression or highest targetknockdown.Panel C: GAPDH activity in Negative Control #1siRNA-transfected cultures, relative to that in the culturetransfected with 0.15 µL of transfection agent; a measure oftransfection-associated toxicity. Arrow indicates transfectioncondition resulting in highest relative GAPDH activity, indicatinglowest cellular toxicity.Panel D: Optimal Balance Factor (OBF)calculated for each transfection condition. Arrow indicates theoptimal transfection conditions (maximum OBF) for this cellline.
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18 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit Data interpretation
Determining optimal transfection conditions using colorimetricdata
Calculating GAPDH activity using colorimetric data
During the assay, GAPDH activity causes a reduction in A615 ofthe samples, although they may eventually acquire a magenta color.For the colorimetric data, subtract the A615 for each sample(A615-sample) from the A615 of the water + Master Mix control(A615-WMM) to determine a GAPDH activity, ΔA615-sample, for thatsample:
For example, the GAPDH activity ΔA615-Neg is obtained bysubtracting A615-WMM from A615 for negative controlsiRNA-transfected cells (A615-Neg):
Construction of the GAPDH Enzyme standard curve
A graph of ΔA615, calculated as described above, for each enzymedilution should yield a line with a positive slope similar to thatyielded by fluorescence data, for example, Figure 3.
Calculating % remaining gene expression and % knockdown of GAPDHexpression
Knockdown is calculated in the same way as for thefluorescence-based assay, once the GAPDH activity, ΔA615, iscalculated. For each transfection condition, the % remainingexpression and % knockdown is calculated from the ratio of ΔA615for samples transfected with GAPDH siRNA to ΔA615 for samplestransfected with negative control siRNA, as follows:
Measuring transfection associated toxicity
For each transfection condition, calculate the GAPDH activity,ΔA615, as described above. The comparison of ΔA615 is similar tothat for the fluorescence-based assay: in samples transfected withnegative control siRNA, the higher the GAPDH activity (ΔA615-Neg),the less toxic the transfection conditions.
Calculating the Optimal Balance Factor with colorimetricdata
The OBF for each transfection condition is calculated withcolorimetric data in the same way as for fluorescence data.
ΔA615-sample = A615-WMM – A615-sample
ΔA615-Neg = A615-WMM – A615-Neg
% remaining expression 100 x ΔA615-GAPDH
ΔA615-Neg-----------------------------=
% knockdown 100 100 x ΔA615-GAPDH
ΔA615-Neg-----------------------------⎝ ⎠
⎛ ⎞–=
OBF ΔA615-Neg % knockdown×=
ΔA615-Neg 100 100 x ΔA615-GAPDH
ΔA615-Neg-----------------------------⎝ ⎠
⎛ ⎞–×=
19KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay KitTroubleshooting
Troubleshooting
General troubleshooting
Visual indicator of GAPDH activity in samples
A visual indicator of GAPDH activity is the magenta coloracquired by some samples after 15 minutes of reaction time. Some ofthe standard curve reactions should turn magenta after 15 minutes.This visible indicator can be used as a double-check for evidenceof GAPDH activity.
No GAPDH activity in standard curve dilutions
If none of the standard curve samples turn acquire a magentacolor after 15 minutes, there was most likely an error in preparingthe KDalert™ Master Mix. We suggest repeating the GAPDH Enzymedilution standard curve reactions.
Low GAPDH activity seen in cultures transfected with thenegative control siRNA or nontransfected
If the standard curve produces the expected result, but lysatefrom cultures there is probably not enough GAPDH in the cellextract used in the assay.
• For the fluorescence assay:– Increase the amount of cellextract to 20 µL per reaction (see “Alternate
procedures” on page 13). – If fewer than 2,000 cells were platedper well, decrease the volume of
KDalert™ Lysis Buffer used to lyse the cells step 2. on page 12proportionately.
• For the colorimetric assay: If fewer than 4,000 cells wereplated per well, decrease the volume of KDalert™ Lysis Buffer usedto lyse the cells proportionately.
No GAPDH gene knockdown
If the standard curve produces the expected result, and all celllysates show GAPDH activity, even those from cultures transfectedwith the GAPDH siRNA. There may have been have from samples withcell extract turn magenta:
• Poor siRNA transfection.• Too much cell extract used inreaction; this is indicated when reactions from
negative control-transfected samples turn magenta in
20 KDalert™ GAPDH Assay Kit User Guide
KDalert™ GAPDH Assay Kit Troubleshooting
A
21KDalert™ GAPDH Assay Kit User Guide
Supplemental information
Related products available from Life Technologies
Quality control
Functional testing A GAPDH Enzyme standard curve with an R-valueof >0.94 was obtained.
Silencer® Select GAPDH siRNAs
Cat. nos. 4390849 (5 nmol) and 4390850 (40 nmol) (Human, Mouse,& Rat)
Silencer® Select GAPDH siRNAs are ideal for developing andoptimizing siRNA transfection conditions. They can also be used asa control in siRNA experiments to confirm that the transfectionprocedure and cell cultures support gene silencing. Silencer®Select GAPDH siRNA (Human, Mouse, & Rat) has been successfullyused in multiple human, mouse and rat cell lines.
Silencer® Select Negative Control siRNAs
Cat. nos. 4390843 or 4390846 (5nmol) and 4390844 or4390847 (40nmol)
Life Technologies has designed and tested two Negative ControlsiRNAs that have no significant sequence similarity to mouse, rat,or human gene sequences. The Negative Control siRNAs are ideal foruse in any siRNA experiment as controls for nonspecific effects ongene expression.
KDalert™ Lysis Buffer
Cat. no. AM8790G
KDalert™ Lysis Buffer provides a one-step lysis and assay bufferfor glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymeactivity in cell extracts.
Lipofectamine® RNAiMAX Transfection Reagent
Cat. nos. 13778-100 (0.1 ml), 13778-03 (0.3 ml), 13778-075(0.75ml) and 13778-150 (1.5 ml)
Lipofectamine® RNAiMAX Transfection Reagent provides the highesttransfection efficiencies on the widest variety of cell types forsiRNA-mediated gene knockdown experiments. Lipofectamine® RNAiMAXis a proprietary RNAi-specific cationic lipid formulation designedspecifically for the delivery of siRNA and miRNA into all celltypes.
22 KDalert™ GAPDH Assay Kit User Guide
AppendixA Supplemental informationQuality controlA
B
23KDalert™ GAPDH Assay Kit User Guide
Safety
WARNING! GENERAL SAFETY. Using this product in a manner notspecified in the user documentation may result in personal injuryor damage to the instrument or device. Ensure that anyone usingthis product has received instructions in general safety practicesfor laboratories and the safety information provided in thisdocument.· Before using an instrument or device, read andunderstand the safety
information provided in the user documentation provided by themanufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicableSafety Data Sheets (SDSs) and use appropriate personal protectiveequipment (gloves, gowns, eye protection, etc). To obtain SDSs, seethe “Documentation and Support” section in this document.
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensurelaboratory personnel read and practice the general safetyguidelines for chemical usage, storage, and waste provided below,and consult the relevant SDS for specific precautions andinstructions:Read and understand the Safety Data Sheets (SDSs)provided by the chemical manufacturer before you store, handle, orwork with any chemicals or hazardous materials. To obtain SDSs, seethe “Documentation and Support” section in this document.· Minimizecontact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses,gloves, or protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemicalcontainers open. Use only with adequate ventilation (for example,fume hood).
· Check regularly for chemical leaks or spills. If a leak orspill occurs, follow the manufacturer's cleanup procedures asrecommended in the SDS.
· Handle chemical wastes in a fume hood.· Ensure use of primaryand secondary waste containers. (A primary waste
container holds the immediate waste. A secondary containercontains spills or leaks from the primary container. Bothcontainers must be compatible with the waste material and meetfederal, state, and local requirements for container storage.).
· After emptying a waste container, seal it with the capprovided.· Characterize (by analysis if necessary) the wastegenerated by the particular
applications, reagents, and substrates used in your laboratory.·Ensure that the waste is stored, transferred, transported, anddisposed of
24 KDalert™ GAPDH Assay Kit User Guide
AppendixB SafetyBiological hazard safetyB
according to all local, state/provincial, and/or nationalregulations.· IMPORTANT! Radioactive or biohazardous materials mayrequire special
handling, and disposal limitations may apply.
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used onthis instrument, the surface may be considered a biohazard. Useappropriate decontamination methods when working withbiohazards.
WARNING! BIOHAZARD. Biological samples such as tissues, bodyfluids, infectious agents, and blood of humans and other animalshave the potential to transmit infectious diseases. All work shouldbe conducted in properly equipped facilities using the appropriatesafety equipment (for example, physical containment devices).Safety equipment also may include items for personal protection,such as gloves, coats, gowns, shoe covers, boots, respirators, faceshields, safety glasses, or goggles. Individuals should be trainedaccording to applicable regulatory and company/institutionrequirements before working with potentially biohazardousmaterials. Follow all applicable local, state/provincial, and/ornational regulations. The following references provide generalguidelines when handling biological samples in laboratoryenvironment. · U.S. Department of Health and Human Services,Biosafety in
Microbiological and Biomedical Laboratories (BMBL), 5th Edition,HHSPublication No. (CDC) 21-1112, Revised December 2009; foundat:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf.
· World Health Organization, Laboratory Biosafety Manual, 3rdEdition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf.
http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdfhttp://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
25KDalert™ GAPDH Assay Kit User Guide
Documentation and support
Obtaining SDSs
Safety Data Sheets (SDSs) are available fromwww.lifetechnologies.com/support.
For the SDSs of chemicals not distributed by Life Technologies,contact the chemical manufacturer.
Obtaining support
For the latest services and support information for alllocations, go to:
www.lifetechnologies.com/support
At the website, you can:
• Access worldwide telephone and fax numbers to contactTechnical Support and Sales facilities
• Search through frequently asked questions (FAQs)• Submit aquestion directly to Technical Support• Search for user documents,SDSs, vector maps and sequences, application notes,
formulations, handbooks, certificates of analysis, citations,and other product support documents
• Obtain information about customer training• Download softwareupdates and patches
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warranttheir products as set forth in the Life Technologies' General Termsand Conditions of Sale found on Life Technologies’ website atwww.lifetechnologies.com/termsandconditions. If you have anyquestions, please contact Life Technologies atwww.lifetechnologies.com/support.
http://:www.lifetechnologies.com/termsandconditionshttp://www.lifetechnologies.com/supporthttp://www.lifetechnologies.com/supporthttp://www.lifetechnologies.com/supporthttp://www.lifetechnologies.com/supporthttp://www.lifetechnologies.com/supporthttp://www.lifetechnologies.com/support
26 KDalert™ GAPDH Assay Kit User Guide
Documentation and support Limited product warranty
Headquarters5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone+1 760 603 7200 | Toll Free in USA 800 955 6288For support visitlifetechnologies.com/support or email [emailprotected]
lifetechnologies.com
May 2013
http://www.lifetechnologies.comhttp://www.lifetechnologies.com/supportmailto:[emailprotected]
KDalert™ GAPDH Assay Kit User GuideCopyrightpageContentsKDalert™ GAPDH Assay KitIntroductionProductdescriptionProcedure overview
Reagents provided with the kit and storage conditionsMaterialsnot provided with the kitKDalert™ GAPDH Assay procedureBefore youstartKDalert™ standard fluorescence procedureAlternateprocedures
Data interpretationDetermining optimal transfection conditionsusing fluorescence dataDetermining optimal transfection conditionsusing colorimetric data
TroubleshootingGeneral troubleshootingFluorescence assay
AppendixA Supplemental informationRelated productsavailable from Life TechnologiesQuality controlFunctionaltesting
AppendixB SafetyChemical safetyBiological hazardsafety
Documentation and supportObtaining SDSsObtaining supportLimitedproduct warrantyBack cover
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