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View additional product information for TaqMan™ Gene Expression Cells-to-CT™ Kit - FAQs (AM1729, AM1728, 4399002)
60 product FAQs found
如果在没有反转录的阴性对照反应中出现PCR产物，而非无模板对照中，则表示RNA样品中仍存在基因组DNA，并且在实时PCR中扩增了基因组DNA。请参考以下建议：
•确保DNase I完全混入裂解液中。
•减少每次裂解反应的细胞用量。
•使用室温下的裂解液裂解细胞，确保裂解反应在室温下发生。
也可尝试延长裂解反应的孵育时间至8分钟，和/或使用已经升温至25°C的裂解液裂解细胞。
在无模板PCR对照中出现PCR产物，表示存在DNA污染。为控制污染，需要采取更为严格的步骤。
请查看以下可能原因和建议：
•加入和混合终止液出现问题：确保将终止液直接加入到裂解物中。如果裂解液的成分未完全灭活，可能会抑制RT-PCR。
•RNA发生降解：开始细胞裂解步骤前，将细胞保存在PBS中并置于冰上。
•样品中的RNase未完全灭活：可能使用了过多细胞或细胞中加入过多PBS，稀释了裂解液。
•样品在恢复到室温前放置太久：加入终止液后，不要将裂解物放置在室温超过20分钟。
•样品不含目标RNA：通过使用样品中的XenoRNAControl确认操作流程是否正常。同时，应确认PCR引物能够在您所使用的PCR条件下扩增目标分子。
可以，该产品具有1mL分装形式（货号 4402960）。
1.确保从孔中移去了所有培养基。
2.移去培养基后，使用相同体积的室温1X PBS洗涤。
3.确保反应在室温下发生（如果将反应板置于冰上、将反应板快速转移至实验台或使用了冰冷的裂解液，则裂解反应可能不会达到室温）。
4.将裂解液恢复至室温后，再加入到细胞。
5.使裂解反应在25°C下持续8分钟。
尽管我们尚未进行测试，但以下文章将我们的试剂盒用于丙型肝炎病毒感染的细胞：
Triyatni M, Berger EA, Saunier B (2011) A new model to produce infectious hepatitis C virus without the replication requirement. PLoS Pathog 7(4)::e1001333.
TaqMan基因表达Cells-to-CT试剂盒可容纳的细胞裂解液体积为逆转录酶反应总体积的45%。
1.加入终止液后，裂解液需在室温下孵育2分钟后再进行后续反应。但是裂解液不能在室温存放超过20分钟。裂解液可以在冰上保存最多2小时，在-20度或者-80度保存不超过5个月。
2.反转录步骤：反转录体系配好后，将体系轻弹混匀，短暂离心将混合好的试剂收集到反应管的底部，反转录反应液可在冰上存放最多4小时。
3.反转录反应完成后，反转录产物可以保存在-20度。
不相同。但是SYBR Cells-to-CT的逆转录酶可以与TaqMan预混液一起使用（反之则不可行）。
有，实时PCR分析结果显示使用Power SYBR Green Cells-to-CT试剂盒和快速SYBR Green Cells-to-CT试剂盒与传统RNA纯化方法提取的RNA结果是有可比性的。大量目标基因的实验结果表明两种试剂盒的检测结果和纯化的RNA结果相当。点击此处(https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/using-sybr-green-for-real-time-rt-pcr.html)，阅读关于SYBR Green Cells-to-CT试剂盒的更多信息。
可以，Cells-to-CT试剂盒可以与TaqMan低密度芯片一起使用。
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。
可以，使用我们的Cells-to-CT试剂盒可以省去RNA纯化步骤。
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。
Cells-to-CT技术的特色是在裂解培养细胞的同时，能够去除基因组DNA并保护RNA完整性。因此，如果您遵循实验方案中的裂解/DNase处理步骤，则无需担心qRT-PCR中存在基因组DNA污染。
可以。Cells-to-CT试剂盒可兼容多重qRT-PCR Assay。如果检测大量靶标，我们推荐结合TaqMan Assay一起使用。
我们尚未将该试剂盒用于植物组织。处理植物样品时，我们建议使用以下任意一种标准匀浆方法：(1)机械化匀浆机，(2)不锈钢珠打磨，或(3)使用研钵和研杵在液氮中研磨组织。
If PCR products are seen in the minus-RT control reaction, but not in the no-template control, it indicates that genomic DNA remains in the sample and that genomic DNA was amplified in real-time PCR. Please follow the suggestions below:
- Ensure the DNase I is mixed thoroughly into the Lysis Solution.
- Use fewer cells per lysis reaction.
- Lyse cells using Lysis Solution that is at room temperature, and make sure that the lysis reaction occurs at room temperature.
You can also try increasing the incubation time of the lysis reaction to 8 minutes and/or using Lysis Solution that has been warmed up to 25 degrees C for cell lysis.
PCR products in the no-template PCR control indicate that the sample is contaminated with DNA. More stringent steps need to be taken to control contamination.
Please review the following possibilities and suggestions:
- A problem with adding or mixing the Stop Solution: ensure that the Stop Solution was added directly to the lysate, as components of the Lysis Solution may inhibit RT-PCR if not fully inactivated.
- The RNA was degraded: keep cells in PBS on ice before starting the cell lysis procedure.
- RNase in the sample was not completely inactivated: Too many cells could have been used or too much PBS left on the cells, diluting the lysis solution.
- The lysates sat too long before going to room temperature: Do not allow lysates to sit longer than 20 minutes at room temperature once the Stop Solution has been added.
- The sample does not contain the target RNA: Verify that the procedure is working by using the XenoRNA Control in the sample. Also check that your PCR primers can amplify your target under the PCR conditions you are using.
Yes, it is available in 1 mL aliquots (Cat. No. 4402960).
1. Ensure that all medium is removed from the wells.
2. Wash with an equal volume of room temperature 1X PBS after the medium is removed.
3. Ensure that the reaction happens at room temperature (the lysis reaction may not reach room temperature if the plate is on ice, if the plate was quickly moved to the bench, or if a cold lysis solution was added).
4. Warm lysis solution to room temperature before adding to cells.
5. Allow the lysis reaction to proceed for 8 minutes at 25 degrees C.
While we have not tested this in house, the following paper did use our kit on cells infected with hepatitis C virus:
Triyatni M, Berger EA, Saunier B (2011) A new model to produce infectious hepatitis C virus without the replication requirement. PLoS Pathog 7(4)::e1001333.
The TaqMan Gene Expression Cells-to-CT Kit can accommodate 45% of the total reverse transcriptase reaction volume as cell lysate.
Here are the potential stopping points:
- At the Cell Lysis step after addition of the Stop Solution and incubating for 2 minutes at room temperature. Do not allow lysates to remain at room temperature for longer than 20 minutes after adding the Stop Solution. Lysates can be stored on ice for a maximum of 2 hours, or at -20 degrees C or -80 degrees C for a maximum of 5 months.
- At the Reverse Transcription (RT) step after assembling the RT reaction. Once assembled, mix reactions gently, then centrifuge briefly to collect the contents at the bottom of the reaction vessel, and store at 4 degrees C for up to 4 hours.
- Completed RT reactions may be stored at -20 degrees C.
No. However, the SYBR Cells-to-CT reverse transcriptase will work with the TaqMan mastermixes (but not the other way around).
Yes, both the Power SYBR Green Cells-to-CT Kit and the Fast SYBR Green Cells-to-CT Kit were compared to a traditional RNA purification method followed by real-time PCR analysis. Both kits show equivalent performance to results obtained using purified RNA over a broad set of gene targets. Read more about the SYBR Green Cells-to-CT kits here (https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/using-sybr-green-for-real-time-rt-pcr.html).
Yes, Cells-to-CT kits can be used with TaqMan Low Density Arrays. Here is a poster showing the workflow with miRNAs: http://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/DNARNAPurification/Files/0714/AACR%20C2CT%20miRNA%20Final.pdf.
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 not had any significant effect on gene expression data.
Yes, we offer our Cells-to-CT kits that allow you to bypass RNA purification.
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)
Cells-to-CT kits were not designed for RNA analysis from bacteria. Some customers do lysozyme incubation before the Cells-to-CT reaction, but the RNA profile could change as a result of this.
Some customers have isolated RNA from viruses. Unfortunately, the protein coat could cause problems. Another option would be to use the MagMax Viral Isolation Kit.
We don't normally monitor RNA quality for Cells-to-CT or any other lysis based methods. We rely on the qRT-PCR results. We have isolated RNA after the lysis reactions and seen that there is no RNA degradation.
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.
In order to monitor for RT inhibition, we recommend using the Cells-to-CT Control Kit (Cat. Nos. 4402959, 4386995). This kit includes an exogenous RNA that is not like any RNA in the databases. Adding this to the RT reaction allows you to monitor for inhibition.
Some customers have used Cells-to-CT kits for small samples, like drosophila eyeballs. Howeveer, Cells-to-CT kits were not designed for such applications.
Cells-to-CT kits can be used to analyze both RNA and DNA. DNA is stabilized as well as the RNA. When doing the analysis, be sure to use a primer and probe set that crosses the exon:intron boundary when you are looking at the RNA.
Unforutnately, the Blood-to-CT and MagMAX kits cannot be used with blood from heparin- or EDTA-stabilized tubes. The reactions require the lysis reagent found in Tempus tubes. Also, the heparin- or EDTA-stabilized tubes don't stabilize the RNA profiles, so we recommend using Tempus tubes.
Yes, Cells-to-CT kits are great for automation, including the screening of siRNAs or small molecules.
We use multiple cells lines in-house, including primary human hepatocytes, stem cells, and differentiated stem cells. When using a new cell line, we recommend that a pilot experiment with a dilution of cells from 10-100,000 cells looking for a loss in linearity of results. Please refer to the manual for how to perform this experiment.
Yes, they are stable at -20 or -80 degrees C for up to 5 months and can be freeze/thawed several times without loss in performance.
For the Cells-to-CT lysis reaction, you can use fresh cells, frozen cells, or cells that have been stabilized with RNAlater solution. You just want to ensure that cells were washed once with PBS before going into the lysis reaction.
The 50 µL Cells-to-CT lysis reaction can be done in any tube or any size plate. If you are using a 6- or 24- well plate, you may need to scale up the lysis reaction to cover the bottom of the plate to lyse all the cells. Also, do not go under the 50 µL reaction volume because the variability when pipetting the stop solution could cause variability with results.
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.
Cells-to-CT technology features a unique method for lysing cultured cells while removing genomic DNA (gDNA) and preserving RNA integrity. Therefore, by following the lysis/DNase treatment steps in the protocol, gDNA contamination will not be an issue for qRT-PCR.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
Yes. Cells-to-CT kits are compatible with multiplex qRT-PCR assays. If a large number of targets are being tested, we recommend combining it with TaqMan assays.
We have not tested plant tissue internally. When working with plants, we suggest employing one of the following standard homogenization methods: (1) motorized homogenizer, (2) stainless steel bead beating, or (3) liquid nitrogen-mortar/pestle.
Please see the following links for Cells-to-CT Stop Solution and Cells-to-CT Bulk Lysis Reagents, which includes the lysis solution, stop solution, and DNase I. The DNase I is not sold separately.
Find additional tips, troubleshooting help, and resources within our Gene Expression Analysis & Genotyping Support Center.
Yes, the Stop Solution provided in the 2-step Cells-to-CT kits contains RNase inhibitor.
The TaqMan Gene Expression Cells-to-CT kit has been validated for duplexing. If you want to set up a multiplex real-time PCR reaction with 3 assays, we recommend using the TaqMan Fast Advanced Cells-to-CT kit (https://www.thermofisher.com/order/catalog/product/A35374).
To prevent signal from genomic DNA in the Cells-to-CT real-time PCR reaction, we recommend using a TaqMan assay or primer set that spans an exon-exon boundary, and adding DNase I to degrade genomic DNA during the lysis reaction. For optimal DNase activity in the lysis reaction, we recommend the following:
1. Ensure all media is removed from the cells.
2. Wash each well or cell pellet with an equal volume of room temperature 1X PBS.
3. Ensure the lysis reaction happens at room temperature. The lysis reaction may not reach room temperature if the plate is on ice prior to adding Lysis Solution, or cold Lysis Solution is added.
4. Warm the Lysis Solution to room temperature before adding to the cells.
5. Perform the lysis reaction at 25 degrees C for up to 8 minutes.
The Cells-to-CT Stop Solution prevents the DNase from being active, even if you add more. If you need to perform additional DNase treatment of the cell lysate sample after the Stop Solution is added, we recommend purifying the RNA from the cell lysate using traditional methods and DNase-treating the purified RNA.
Yes, the TaqMan Fast Advanced Master Mix (Cat. No. 4444557) can be used in place of the TaqMan Gene Expression Master Mix (Cat. No. 4369016) when setting up the qPCR reaction for the TaqMan Gene Expression Cells-to-CT kit.
We have not tested the compatibility of Cells-to-CT kits with plasma samples and would not recommend using this kit for plasma samples. Please refer to the following table to select an RNA purification kit based on your sample type: https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-types/total-rna-extraction.html.
The following 2-step Cells-to-CT kits use a mixture of oligo dT and random primers for the reverse transcription step:
• TaqMan Fast Advanced Cells-to-CT Kit
• TaqMan Gene Expression Cells-to-CT Kit
• SYBR Green Fast Advanced Cells-to-CT Kit
• Power SYBR Green Cells-to-CT Kit
• Fast SYBR Green Cells-to-CT Kit
The following 1-step Cells-to CT kits require gene-specific primers for the reverse transcription step, so you can either use your SYBR qPCR primers or TaqMan assay primers:
• Cells-to-CT 1-Step TaqMan Kit
• Cells-to-CT 1-Step Power SYBR Green Kit
We do not recommend scaling down the lysis reaction volume for the Cells-to-CT kits. Reducing the lysis reaction volume below 50 µL can lead to incomplete inactivation of reagents and cause variability with results.
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