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What options do you offer for luciferase vectors? Product FAQ

Answer

There are three vectors available for each expressed luciferase; pMCS, pCMV, and pTK. We recommend using pMCS to measure the activity of the reporter, and pCMV, and pTK as positive control vectors. For high level constitutive expression of Luciferase, we recommend using the CMV (Cytomegalovirus) promoter vector and for low level constitutive expression of luciferase, we recommend using the pTK (thymidine kinase) promoter vector.

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What tests did you perform to demonstrate increased protein expression levels with pCDNA4/HisMax-TOPO vector? Product FAQ

Answer

Recombinant GFP, LacZ, and Luciferase proteins were cloned into pcDNA4/HisMax-TOPO vector and transiently transfected into 5 cell lines: CHO, COS-1, NIH-3T3, HeLa, and 293. After 48-72 hours, samples were analyzed by western blot. In all cases, expression levels of the proteins were found to be 2 to 5-fold higher than the levels obtained with a similar vector lacking the QBI SP163 element. In COS-1 cells, measurement of GFP fluorescence also showed a more than five-fold increase over the samples from the control vector.

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What controls and control siRNAs do I need? Product FAQ

Answer

Good transfection is absolutely essential for effective target knockdown using siRNA, thus it is important to include a positive control siRNA in each experiment. The positive control siRNA should elicit reproducible, easily measured knockdown and/or phenotype in the cells and assay used in your study. If you see maximal knockdown or a phenotype above/below a pre-determined threshold level with this control, you know that measurements from other siRNAs tested on the same day are reliable. Note that it is important to empirically determine the thresholds for each assay and siRNA control pair that indicate a good transfection.

In siRNA experiments, negative controls are just as important as positive controls for obtaining meaningful data. Always include a set of transfections with an equimolar amount of at least one non-targeting negative control siRNA (e.g., Invitrogen Silencer Select Negative Control #1) - data from these crucial controls serve as a baseline for evaluation of experimental target knockdown.

Nontransfected or cells-only negative controls are also very useful in siRNA experiments. By comparing expression of a housekeeping gene among cultures that were not transfected and cultures transfected with a non-targeting negative control siRNA, valuable information about the effects of transfection on cell viability can be obtained.

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The Importance of Temperature Control with Abbe Refractometers Product Literature

What is your siRNA guarantee? Product FAQ

Answer

Please see the following:

(a) Silencer Select siRNA: “Thermo Fisher Scientific guarantees that when you purchase two Silencer Select Pre-designed siRNA to the same target, then those two siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ?5 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer Select siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product.”
(b) Stealth siRNA: “Thermo Fisher Scientific guarantees that when you purchase three Stealth Pre-designed siRNA to the same target, then at least two of those three independent, nonoverlapping siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ?20 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product. We also recommend the use of an appropriate negative control, such as one of the three Stealth RNAi Negative Controls, to normalize message knockdown.”
(c) Silencer siRNA: “Thermo Fisher Scientific guarantees that when you purchase three Silencer Pre-designed siRNAs to the same target, at least two of the siRNAs will reduce target mRNA levels in cultured cells by 70% or more when measured 48 hours after transfection at 100 nM or higher final siRNA concentration under the conditions described below. If at least two of the three siRNAs do not induce >70% target mRNA knockdown, Thermo Fisher Scientific will provide a one-time replacement of up to three Silencer Pre-designed siRNAs per target at no additional charge. Requests for replacement product must be made within one hundred and eighty (180) days from the date of delivery of the Silencer Pre-designed siRNAs. Optimum transfection efficiency must be confirmed using good laboratory practices and a proven-to-work siRNA to an endogenous message, such as Invitrogen Silencer GAPDH siRNA Control. To assess knockdown, target mRNA levels in treated samples must be compared to that of cells transfected with a nontargeting control siRNA, such as Silencer Negative Control #1. We recommend Applied Biosystems TaqMan Gene Expression Assays to quantify mRNA levels.”

Answer Id:: E9886

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Is there a way for me to screen sequences to determine if they work for RNAi knockdown? Product FAQ

Answer

Yes, we offer a pSCREEN-iT/lacZ-DEST Gateway Vector Kit which can be used to assess the potency of any RNAi knockdown without knowing protein function, western blotting or qRT-PCR. The system uses a beta-galactosidase activity assay to measure the knockdown ability of an RNAi reagent (Stealth siRNA, Silencer siRNA, shRNA-containing plasmid, Dicer pools, etc). Clone your target gene into the vector provided, and co-transfect it along with the knockdown reagent being tested. Expressed genes will be fused to beta-galactosidase, enabling you to use beta-Gal levels to measure the amount of RNAi-induced degradation of the target gene.

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Should I run any internal controls with your ELISA kits? Where can I get internal controls? Product FAQ

Answer

Running internal controls along with the blanks and the standard curve is an excellent way to monitor the performance of any ELISA kit. Internal controls are samples containing known amounts of the analyte being measured with the kit. A key attribute of internal controls, and what differentiates them from the samples of the standard curve, is that they duplicate the composition of your actual samples as closely as possible. This is important because your samples may contain components that affect detection of the analyte differently than does the Standard Diluent provided in the kit. In other words, internal controls allow you to determine if assaying the same amount of analyte in the Standard Diluent (the standard curve) and in the sample matrix (internal controls) gives the same results. It's also important to remember that internal controls give you confidence in the accuracy of your results. Internal controls help ensure that the assay is performing consistently from assay to assay, every time you run it.

One source of internal controls is naturally occurring samples (such as serum or plasma) containing known amounts of the analyte you want to measure. Such positive control samples can be run as-is, or they can be diluted to various known concentrations, preferably with a naturally occurring negative sample of the same type. It's a good idea to run at least 2 internal controls, if you can. One usually has an analyte concentration between the lowest point on the standard curve and the curve's midpoint, while the other has a concentration between the midpoint and the highest concentration. Some researchers also run an internal control that falls close to the midpoint of the curve.

If you don't have access to naturally occurring controls, you can prepare them yourself. Let's say that you will be measuring interleukin-6 (IL-6) in human serum samples with an ELISA kit, Cat. No. KHC0061. First you will need to obtain some pooled normal human sera, which will be used as the sample matrix for your internal controls. Preferably, the IL-6 content of this serum should be negligible (but known) or too low to measure. Next you should add known amounts of human IL-6 to this serum to create the internal controls, using the human IL-6 standard provided in the kit. After you prepare the standard curve as described in the product manual, add some of the leftover concentrated IL-6 standard to the serum matrix. As described above, you can prepare controls with high and low IL-6 levels, or high, medium, and low, if you have enough wells in your ELISA plate.

Even if your sample type is not serum and you're not measuring IL-6, follow the general procedure outlined above to prepare your internal controls with your protein and sample matrix of interest. A valuable resource describing Spike and Recovery and Linearity of Dilution assays can be found here (http://tools.thermofisher.com/content/sfs/brochures/TR0058-Spike-and-Recovery.pdf). If you need more help, please contact Technical Support at techsupport@thermofisher.com.

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How can I check that my protein or peptide was reduced efficiently using the Immobilized TCEP Disulfide Reducing Gel? Product FAQ

Answer

There are several ways to assess the level of sample reduction. Each method expends a small amount of sample. Ellman's Reagent (Cat. No. 22582) allows colorimetric determination of free sulfhydryl content by comparison to cysteine (Cat. No. 44889) standards. If appropriate control lanes are included in the gel, sample reduction can be assessed by SDS-PAGE using non-reducing conditions. After capping the free sulfhydryls by alkylation, N-ethylmaleimide (NEM, Cat. No. 23030), or cyanylation (1-cyano-4-dimethylamino-pyridinium tetrafluoroborate, CDAP), HPLC may also be used to measure sample reduction.

Answer Id:: E8579

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I am trying to target a big and multifunctional protein using CRISPR technology. To what site should I design my sgRNA? How can I check that the editing occurred? Product FAQ

Answer

The first few exons would be best (closer to the promoter, resulting in premature transcript termination). Since the gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location, it is advisable to design and test a few target sites. Non-CRISPR-related mutations may be identified using gDNA isolated from non-CRISPR-treated cells as a control and performing a Invitrogen GeneArt Genomic Cleavage Detection Assay (https://www.thermofisher.com/order/catalog/product/A24372). Standard western blot analysis is a good measure for the verification of protein levels.

Answer Id:: E10128

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What endogenous control to use for non-human and non-mouse miRNA assays? Product FAQ

Answer

It is important to select the appropriate endogenous control to normalize your data. Thermo Fisher Scientific has identified a number of small non-coding RNAs (snoRNAs, snRNAs) that show stable, moderate expression across a large number of tissues for human and mouse that are therefore good candidates for an endogenous control. Candidate controls are also available for rat, Drosophila, Arabidopsis and C. elegans. An updated list of available controls can be found on our portal under the URL www.thermofisher.com/taqmanmirna by selecting MicroRNA and then Controls, followed by the species of interest as search options.

In general, any miRNA can be used as an endogenous control to normalize results across different samples as long as it meets the criteria of a good endogenous control: providing stable expression levels with minimal variation across the different sample and conditions being used in your study. Since a control for one experimental study/treatment may not be appropriate for another one, a control should be validated before use. One approach is to select the control(s) based on your data set. An Application Note that describes how to perform validation using simple statistical methods, i.e. by measuring the Standard Deviation of the average Ct, is available: Endogenous Controls for Real-Time Quantitation of miRNA Using TaqMan MicroRNA Assays (Application Note: TaqMan MicroRNA Assays, 2007). Alternatively, you can use geNorm (Vandensompele et al. 2002 Genome Biology, http://genomebiology.com/2002/3/7/research/0034) to determine which of the candidate controls is best to use for your study.

Answer Id:: E7051

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What is the difference between the Cytoscreen ELISA format and the EASIA format? Product FAQ

Answer

Our EASIA (Enzyme Amplified-Sensitivity Immuno Assay) kits are three-member solid-phase sandwich assays, also called direct ELISA kits. They contain 96-well plates coated with one or more F(ab')2 monoclonal capture antibodies. The standards in these kits are provided as lyophilized powders containing analyte at discrete levels plus plasma proteins. Standards are reconstituted by adding water. EASIA kits also include lyophilized controls with known levels of analyte in plasma protein. In performing the EASIA, sample, standard, or control is pipetted onto the plate. Next, horseradish peroxidase (HRP) conjugated detection antibody is added to complete a three-member solid-phase sandwich. Following a washing step, the HRP activity is quantitated by incubation with substrate solution (TMB plus H2O2). At the end of the incubation step, the HRP reaction is terminated by the addition of an acidic stop solution and the optical density of the solution in the wells is measured with a spectrophotometric ELISA plate reader. EASIA kits are validated for measuring human serum, plasma, culture supernatant, and other biological fluids.

Our Cytoscreen ELISA kits also contain pre-coated 96-well plates. The standard curve is constructed by reconstitution and serial dilution of a single vial of standard using standard diluent buffer provided in the kit. After samples or standards are pipetted into the wells, a biotin-labeled detection antibody is added, followed by a wash step and addition of HRP-conjugated streptavidin to complete a four-member solid-phase sandwich. As with the EASIA kits, the HRP activity is quantitated by incubation with substrate solution (TMB plus H2O2) followed by an acidic stop solution. The optical density is measured with a spectrophotometric ELISA plate reader. Cytoscreen ELISA kits are validated for measuring analytes in serum or plasma, culture supernatant, and other buffered solutions, and are available for quantitation of human, mouse, rat, monkey, and pig samples.

Answer Id:: E5149

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What is the lowest cytokine concentration that can be measured with confidence using a cytokine ELISA kit? Product FAQ

Answer

Many physiological samples contain low levels of cytokines. In determining the lowest concentration that can be measured with confidence by an ELISA, there are a couple of specifications to consider:

1) The range of the ELISA kit. A typical range for a cytokine ELISA kit is 0 - 1000 pg/mL. The standard curve for this range would be made up of data points corresponding to 0, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 pg/mL.

2) The reported statistical sensitivity of the kit. This is determined by measuring the OD values of control samples containing zero standard concentration in 30 replicates as well as measuring a standard curve in quadruplicate. The mean OD and standard deviation of these 30 replicates is calculated, and two standard deviations are added to the mean to give the lowest statistically significant result. The corresponding concentration is calculated from the standard curve to give the statistical sensitivity of the ELISA in terms of cytokine concentration.

For a kit with a range of 0 to 1000 pg/mL, a typical statistical sensitivity will usually be on the order of 5 pg/mL. The concentration detected in many physiological samples will fall between the 0 pg/mL and 15.6 pg/mL. As long as the value detected is above the statistical sensitivity of the ELISA, 5 pg/mL or greater in the example case, the value is statistically significant. One cannot be certain about the validity of measurements less than the statistical sensitivity of the kit and should report the corresponding results as below the limit of detection by the kit.

Answer Id:: E5121

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How do I measure the effect of a siRNA? Product FAQ

Answer

The most common way to measure gene specific knockdown is to perform real-time PCR. In some cases a reporter system that allows easy measurement of a reporter gene, such as beta-galactosidase, may be used. Western blot analysis to compare the level of protein expression before and after the introduction of siRNA may also be employed.

Answer Id:: E9897

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Specification Sheet: Mercury Analyzer - 80i Data Sheet Product Literature

What approach should be taken to measure target knockdown after siRNA transfection? Product FAQ

Answer

Measure mRNA levels by real-time PCR. A typical time course analysis would measure at 24, 48, and 72 hours. Protein analysis may be checked at 48, 72, and 96 hours. This will vary depending on the gene being targeted.

Answer Id:: E9913

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