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View additional product information for TaqMan™ SCID/SMA Plus Assay - FAQs (A48568, A48569, A48566, A48567)
51 product FAQs found
The TaqMan® SCID/SMA Plus Assay (Cat. Nos. A48566, A48567, A48568, A48560) uses the following fluorescent reporter dyes: FAM™ dye, JUN™ dye, ABY™ dye, and VIC™ dye.
Please refer to page 16 of the TaqMan® SCID/SMA Plus Assay User Guidefor more information about the reporter dyes used in this assay.
Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.
Please refer to the following TaqMan SCID/SMA Plus Assay User Guide (pg 6) for instructions on ordering plasmids from GeneArt.
No, you will need to order the TREC and KREC plasmids separately. Please refer to the following TaqMan SCID/SMA Plus Assay User Guide (pg 6) for instructions on ordering plasmids from GeneArt.
Yes, the TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay are compatible with QuantStudio 6 and 7 instruments using QuantStudio Software v1.6.0 (or newer) and firmware v1.0.4 (or newer).
The user is responsible for ordering the required calibration plates prior to the onsite training. If you purchase the onsite training, you may ask the FAS to complete the instrument dye calibration.
The NBS training (Cat. No. A48619) is a 1 day onsite FAS training which includes hands-on workflow training.
We cannot provide a specific diagnostic copy number cutoff for SCID.
We recommend using Auto Ct (Auto threshold) and auto baseline until the Ct cutoff for each target has been set through your clinical validation study.
We have not validated detection of XLA with the TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay.
The TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay can be used for semi-quantitative analysis for KREC or TREC targets if you use a standard curve.
No, the TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay are not designed to determine SMN2 copy number.
No, the TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay are not intended to determine SMA carrier status.
SMA testing determines the absence or presence of SMN1. A person who is considered normal has ≥1 copy of SMN1. Conversely, a person with SMA has a homozygous deletion of SMN1. The TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay determine a “Yes/No” result. Presence of SMN1 amplification curves indicates a normal (unaffected) sample, and an absence of SMN1 amplification curves (i.e., undetermined SMN1 Ct) indicates an affected sample.
The TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay are specific to SMN1. SMN2 does not impact SMN1 amplification. The assay has been challenged with high copy number of SMN2 target and it did not affect amplification.
SMA testing determines the absence or presence of SMN1. A person who is considered normal has ≥1 copy of SMN1. Conversely, a person with SMA has a homozygous deletion of SMN1. SMN1 and paralog SMN2 differ by five bases, including a single coding base pair (exon 7 C>T) used to detect the deletion of SMN1 or gene conversion to SMN2. Therefore, specificity of SMN1 is important, as cross hybridization of SMN2 can cause a false positive signal of SMN1 presence. Due to the potential cross hybridization to SMN2 gene targets, we have designed this assay to “block” SMN1 probe cross-hybridization to SMN2 genomic targets, and therefore provide high specificity to SMN1.
SMA testing determines the absence or presence of SMN1. A person who is considered normal has ≥1 copy of SMN1. Conversely, a person with SMA has a homozygous deletion of SMN1. SMN1 and paralog SMN2 differ by five bases, including a single coding base pair (exon 7 C>T) used to detect the deletion of SMN1 or gene conversion to SMN2. Therefore, specificity of SMN1 is important, as cross hybridization of SMN2 can cause a false positive signal of SMN1 presence. Due to the potential cross hybridization to SMN2 gene targets, we have designed this assay to “block” SMN1 probe cross-hybridization to SMN2 genomic targets, and therefore provide high specificity to SMN1.
The exported JSON file should have a .JSON file extension. If the exported JSON file does not have the .JSON extension once it is exported, click export again, but do not change the file name. Save the exported JSON file in its final location and then change the file name. Changing the JSON file name before saving may alter its file extension.
If you are using DA Software v2.4.3 or newer, a JSON file can be created for analysis parameters.
Incorporating a JSON file into DA Software will allow customers to generate reports from test results. Defined parameters for data analysis can be entered into a JSON file. This JSON file can then be applied to each subsequent sample run, thus allowing for quick and consistent reporting of testing results.
The most current version of the DA Software can be downloaded here: https://www.thermofisher.com/us/en/home/global/forms/life-science/quantstudio-6-7-pro-software.html
We recommend using DA Software v2.4.3 or newer. The most current version of the software can be downloaded here: https://www.thermofisher.com/us/en/home/global/forms/life-science/quantstudio-6-7-pro-software.html
The TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay are shipped at ambient temperature (https://assets.thermofisher.com/TFS-Assets/LSG/brochures/cms_081489.pdf) and we recommend storing them at -20 degrees C upon receipt.
Yes, DNA extract obtained from a 1.5 mm punch size can be used on a 384-well format.
The TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay protocol is compatible with all 3 QuantStudio block types: 96-well 0.1 mL, 96-well 0.2 mL, and 384-well.
The TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay have been validated on the QuantStudio 5, QuantStudio 6 Flex, QuantStudio 7 Flex, QuantStudio 12K Flex, and QuantStudio Dx (in RUO mode) instruments. The assays are also compatible with the QuantStudio 6 Pro and QuantStudio 7 Pro instruments but have not been validated on these instruments.
For all QuantStudio instruments, we recommend setting the ramp rate to the default speed.
Yes, standard cycling may be used for the TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay.
Assuming ˜100% PCR efficiency, a 2-fold, 5-fold, or 10-fold serial dilution can be used to generate a standard curve. We recommend using a 10-fold serial dilution of known concentration of target constructs.
We recommend using 4-6 dilution points. Each dilution run in triplicates should suffice.
Yes, since the assay itself is multiplex, a single mix of TREC and KREC plasmids can be used to generate standard curves for TREC and KREC simultaneously. If using a 20 µL reaction volume, use 4.5 µL of each plasmid for a total volume of 9 µL. Adjust the calculated copies/reaction accordingly.
For short term storage, we recommend storing dried blood spots (DBS) desiccated or non-desiccated at room temperature.
For long term storage, we recommend storing dried blood spots (DBS) at room temperature or at -20 degrees C.
Based on our stability study data, the extracted lysate from 3.2 mm DBS samples is stable for up to 12 days at RT and 4 degrees C.
Yes, as long as the pH is 8, regular TE (with 1 mM EDTA) or low EDTA TE (0.1 mM EDTA) can be used to prepare the stabilizing buffer. However, low EDTA TE is preferred to minimize interference with the Mg2+ present in the qPCR reaction.
We recommend using Design and Analysis (DA) Software v2.4.3. or newer for analyzing data from the TaqMan SCID/SMA Assay and TaqMan SCID/SMA Plus Assay. Please download the latest version of the software from here: https://www.thermofisher.com/us/en/home/global/forms/life-science/quantstudio-6-7-pro-software.html
Yes, 0.05% Tween 20 can be used in the stabilizing buffer. We have tested 0%, 0.05%, 0.10%, and 0.5% Tween 20 in the stabilizing buffer, and with all these Tween 20 concentrations, target amplification curves were comparable. At 5% Tween 20 in the stabilizing buffer, SMN1 amplification was affected significantly, while RNaseP and TREC amplification curves were not as affected.
The pipette should be set to above 150 µL to ensure that all liquid (eg., 157 µL) is removed from the punch. Leaving liquid in the punch will have negative consequences and may leave out impurities/interfering substances that can impact the SMN1 curve profile.
The DBS wash buffer will not settle out at RT, so it can be stored.
The Thesit reagent can be warmed up in a water bath until it liquifies.
We recommend adding 0.5 mL Tween 20 to 100 mL TE (0.005X TE volume) or 120 µL Tween to 24 mL TE.
Our protocol is a transfer protocol, so it will always start in a 96-well plate. However, labs can validate their own methods.
According to Centers for Disease Control and Prevention (CDC), 1.5 mm punch size is the most popular in the US.
Yes. Assay performance is comparable if QuantaBio ExtracTa is used instead of 0.5% Thesit wash buffer and Extract All Lysis Solution to extract DNA.
If the Stabilizing Solution from the Extract All Kit is used, target amplification curves will appear S-shaped.
We have tested 0%, 0.5%, and 1.0% Thesit concentration in the DBS wash buffer and have not observed significant effects on the target amplification curves. At 5% Thesit concentration in the DBS wash buffer, SMA assay performance was slightly affected, however, TREC and RNaseP targets amplified well.
There is no significant effect on the target amplification curves with 0% Thesit in the DBS wash buffer.
We have tested 0%, 0.05%, 0.10%, and 0.5% Tween 20 in the stabilizing buffer, and with all these Tween 20 concentrations, target amplification curves were comparable. At 5% Tween 20 in the stabilizing buffer, SMN1 amplification was affected significantly, while RNaseP and TREC amplification curves were not as affected.
Having no Tween 20 in the stabilizing buffer does not significantly affect the target amplification curves.
We have tested 30 min, 1 hr, 2 hr, and 3.5 hr wash duration (on shaker), and we did not observe significant effects on the target amplification with prolonged DBS washing.
For non-desiccated and freshly prepared samples, 10 min of DBS washing with wash buffer works well, however, customer will need to validate this modified workflow.
The validated dried blood spots (DBS) wash protocol includes centrifugation at 1500 rpm for 30 min. This protocol is suitable for various DBS sample types: desiccated, non-desiccated, >7 days from preparation, ≥7 days from preparation (freshly prepared). A higher rpm is required for DBS samples that have been desiccated for long term storage to adequately remove DNA from the DBS punch.
Extracted dried blood spots (DBS) punches obtained from freshly prepared samples appear nearly white/cream. If the DBS sample was prepared >7 days ago or has been desiccated for a prolonged period of time, the color may appear light red/brown. If the extracted DBS punch remains a dark red/brown color, additional wash time may be required.