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View additional product information for Single Cell-to-CT™ qRT-PCR Kit - FAQs (4458237, 4458236)
17 product FAQs found
Cells-to-CT系统应该能适用于所有细胞系。但是，由于细胞大小和成分的差异，每个裂解反应的最大细胞数在不同细胞系中会有轻微差异。您可使用TaqMan对照试剂盒检测抑制和最小样品起始量。
裂解后样品可在室温下保存最多30分钟，加入终止液后可放置多至2小时。您可将细胞裂解液、cDNA和预扩增样品冻存，待后续处理。
在使用Snigle Cell-to-CT试剂盒时，由于单个细胞个体差异以及转录差异的存在，我们不建议使用内参对数据进行归一化处理。由于存在这种差异，归一化反而会人为增加计算出来的单个细胞基因表达水平和实际值的差异。
我们已经测试了将试剂反复冻融5-10次，发现对CT值无影响。裂解样品冻融多达5次，对基因表达数据无任何显著影响。
是的，使用对Megaplex Pool进行了修改的用于MicroRNA表达分析的操作方案(P/N 4399721 Rev. B)。在本方案中，对逆转录和预扩增反应进行体系放大以包括所有细胞裂解物。
If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:
- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript VILO Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT type workflow (depending on your sample type)
With very small samples, it is always better to use a lysis-based solution so that you don't lose any of your sample. Cells-to-CT kits, for example, can accommodate as few as 10 cells. If your sample size is smaller than 10 cells, we recommend using the Single Cell-to-CT Kit.
There is no reason why the Cells-to-CT system shouldn’t work with any cell line. However, due to differences in cell size and composition, the maximum number of cells per lysis reaction may be slightly different for different cell lines. We recommend testing for inhibition and optimal cell input by using the TaqMan Cells-to-CT and SYBR Green Cells-to-CT Control kits.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
Samples can be placed at room temperature for up to 30 minutes following lysis, and for up to 2 hours after adding Stop Solution. Cell lysates, cDNA, and preamplification samples can all be frozen for future processing.
We do not recommend normalizing to an endogenous control due to the biological variation and transcriptional noise exhibited by single cells. Because of this variation, normalization can actually increase the spread of calculated expression levels in single cells.
We have tested the reagents that are stored frozen with five to ten freeze–thaw cycles and have seen no effect on Ct values. Up to five freeze–thaw cycles for the lysate samples have been shown to have no significant effect on gene expression data.
Yes, using a modification of the Megaplex Pools for MicroRNA Expression Analysis Protocol (P/N 4399721 Rev. B). In this protocol, scale the reverse transcription and preamplification reactions to include all of the cell lysate.
This kit will work with sorted cells as long as the volume of the cells is less than 5µl (10% of the lysis volume). Additionally, we recommend sorting the cells with PBS, as media, especially one containing FBS, can inhibit the lysis efficiency. Briefly, harvest cells, wash in PBS, remove the wash and resuspend in PBS to sort.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
While we have not tried this in-house, there is reason to believe this should work. However, optimization of the protocol around the RT step may be needed, as FFPE samples have a lot of RNA crosslinking, which could affect the efficiency of the RT enzyme. Heating the sample for 10 min at 80 degrees C before adding the RT in order to get good cDNA conversion should improve reverse transcription.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
1. Ensure that all media is removed from the wells.
2. Wash with an equal volume of room temperature 1X PBS.
3. Ensure that the lysis reaction happens at room temperature (the lysis reaction may not reach room temperature if the plate is on ice, quickly moved to the bench, or cold lysis solution is added).
4. Warm the lysis solution to room temperature before adding to the cells.
5. Perform lysis reaction at 25 degrees C for up to 8 mins.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
To identify the maximum number of cells to use for each reaction, we recommend testing a range of cellular input amounts by setting up a serial dilution. Instructions for determining the best cell number input with Fast Advanced Cells-to-CT kits can be found in the User Bulletin: Cell Input Optimization for SYBR Green Fast Advanced Cells-to-CT Kit (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017931_CellInputOptimization_SYBRGreenFastAdvCells-to-CT_UB.pdf) and the User Bulletin: Cell input optimization for TaqMan Fast Advanced Cells-to-CT Kit (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017932_CellInputOptimization_TaqManFastAdvCells-to-CT_UB.pdf). For the non-Fast Advanced Cells-to-CT kits, instructions can be found in the Pilot Experiment section of the protocol for each kit.
Here is a short list of cell lines that have been tested with the Cells-to-CT system: HeLa, HepG2, primary hepatocytes, SK-N-AS, SK-N-SH, U-87 MG, ME-180, A549, Jurkat, PC-12, PT-K75, NIH/3T3, Raji, HEK-293, COS-7, CHO-K1, NCI-H460, DU-145, K562, U-2 OS, Huh-7, Neuro 2A, and BJ. For additional information please visit the following page: https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-types/total-rna-extraction/cells-to-ct-kits/cells-to-ct-faq.html