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View additional product information for SYBR™ Green Cells-to-CT™ Control Kit - FAQs (4402959)
41 product FAQs found
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.
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.
Yes. The Cells-to-CT control kits contain Xeno RNA and assays/primers for detecting Xeno RNA and B-actin. They can be used for troubleshooting and setting up a pilot experiment to determine optimal cell input.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications 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.
Yes. The TaqMan Cells-to-CT Control Kit and SYBR Green Cells-to-CT Control Kit are compatible with the Fast Advanced Cells-to-CT kits and workflow.