Invitrogen™ Single Cell-to-CT™ qRT-PCR Kit

Catalog No.	No. of Reactions
44-582-36	400 Reactions
44-582-37	50 Reactions

Catalog No. 44-582-36 Supplier Invitrogen™ Supplier No. 4458236

Description

The Ambion Single Cell-to-C_T™ Kit contains a complete validated workflow for gene expression analysis for samples containing 1–10 cells.

The Ambion Single Cell-to-C_T™ Kit contains a complete validated workflow for gene expression analysis for samples containing 1–10 cells. Each kit contains reagents for sample preparation, reverse transcription, pre-amplification, and qPCR that have been optimized together in a simple workflow that can be completed in only five steps.

Pre-optimized protocol ensures success and saves time

Each workflow begins with a simple 7-minute sample prep, where cells are effectively and reproducibly lysed with minimal processing into a solution that is compatible with downstream RT-PCR, without the need for purification. This is followed by the use of Superscript™ RT for reverse transcription, the TaqMan™ PreAmp Master Mix to amplify the cDNA, and TaqMan Gene Expression Master Mix for qPCR. All four of these steps have been developed to provide maximum sensitivity. The entire cell sample is maintained in the same well throughout the procedure so there is no sample loss or dilution of the precious limited material that could affect sensitivity.

Single cell sensitivity

Sensitivity of real-time PCR results can be affected by the efficiency of sample preparation, reverse transcription, or amplification. The Single Cell-to-C_T Kit has addressed each of these potential problem areas to create a solution that enables reliable and robust gene expression analysis from single cells with maximal sensitivity. Detection of single cell equivalents using the Single Cell-to-C_T Kit demonstrates appropriate linearity and sensitivity of qPCR results compared to a 100 cell sample control with excellent technical reproducibility. As expected, individual single cells exhibit greater variability due to inherent biological heterogeneity. In addition, through inclusion of a cDNA pre-amplification step, targets of interest are accurately amplified prior to real-time PCR. This critical step boosts signal in an unbiased manner, extending the analysis potential of limited samples. The accuracy and signal enhancement of a 96-gene panel using pre-amplification is shown.

Superior performance and reproducibility compared to alternative methods

Traditional sample preparation methods using organic solvents, glass fiber filters, or magnetic beads are not suitable for single-cell analysis due to the loss of sample through incomplete RNA precipitation, binding, and elution. Additionally, most single-cell homebrew methods which involve simple boiling to lyse cells lack the ability to inactivate endogenous RNases to stabilize gene expression profiles and can result in chemical cleavage of the RNA and inhibitor carryover, including gDNA contamination, which can affect all qRT-PCR applications. In contrast, the Single Cell-to-C_T Kit shows superior sensitivity and reproducibility for single-cell analysis compared to traditional purification or homebrew boiling methods.

• Pre-optimized workflow for real-time RT-PCR from single cells
• Maximum sensitivity for single-cell analysis
• Superior performance and reproducibility compared to alternative methods
• Complete kit convenience and easy-to-follow protocol
• Suitable for use with 1-step and 2-step qRT-PCR
• Less hazardous, less waste, fewer resources used

Order Info

Shipping Condition: Dry ice

Specifications

• Content And Storage: 400 μL Single Cell DNase I, 4.0 mL Single Cell Lysis Solution (store at 4°C), 400 μL Single Cell Stop Solution, 600 μL Single Cell SuperScript™ RT, 50 mL TaqMan™ Gene Expression MasterMix (store at 4°C), 2.1 mL Single Cell Pre-Amp Mix, 1.2 mL Single Cell VILO™ RT Mix, Store kit components at -20°C except for Single Cell Stop Solution and TaqMan™ Gene Expression MasterMix, which should be stored at 4°C.
• Detection Method: TaqMan
• PCR Method: 2-step RT-qPCR
• Reaction Speed: 7 min.
• For Use With (Equipment): 7000 System, 7300 System, 7500 System, 7900HT Fast System, 7900HT System, GeneAmp 9700, GeneAmp™ 2400, StepOne™, Fast Mode, StepOne™, Standard Mode, StepOnePlus™, Fast Mode, StepOnePlus™, Standard Mode, Veriti Thermal Cycler
• Product Line: Ambion, Cells-to-CT
• Purification Time: 7 min.
• Quantity: 400 reactions
• Shipping Condition: Dry Ice
• For Use With (Application): RT-PCR
• No. of Reactions: 400 Reactions
• Sample Type: Mammalian Cells
• Starting Material: 1-10 Cells

Frequently Asked Questions (FAQs)

What can I do to improve the sensitivity of my qPCR assay?

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)

Would the direct lysis approach to DNA/RNA analysis be a good alternative for very small samples?

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.

Will the Cells-to-CT system work with my special cell line?

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.

With the Single Cell-to-CT Kit, do samples need to be frozen immediately?

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.

For single-cell analysis using the Single Cell-to-CT Kit, do I need to normalize my data?

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.

Can Single Cell-to-CT kit reagents be subjected to freeze–thaw cycles?

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.

Can miRNAs be detected using the Single Cell-to-CT Kit?

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.

Can cells sorted by flow cytometry be used with the Single Cell-to-CT qRT-PCR Kit (Cat. No. 4458237)?

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.

Can laser micro-dissected FFPE single cells be used with a Single Cell-to-CT Kit?

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.

How do I remove gDNA from my Cells-to-CT reaction?

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.

What is the optimal number of cells to use per reaction with Cells-to-CT kits?

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.

Which cell lines have been tested to work with the Cells-to-CT system?

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

For Research Use Only. Not for use in diagnostic procedures.