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Ambion® Single Cell-to-CT™ Kit for qRT-PCR

  • Maximum sensitivity for single-cell analysis
  • Complete kit convenience and easy-to-follow protocol

This new kit provides a validated workflow for gene expression analysis, and offers a standardized platform for the study of single cells. View the frequently asked questions.

Ambion® Single Cell-to-CT™ Kit for qRT-PCR


The Ambion® Single Cell-to-CT™ kit enables you to study gene expression at the single-cell level, without having to first isolate RNA. The kit is optimized for maximum sensitivity for reliable, consistent results even when starting from a single cell. This new kit provides not only a validated workflow for gene expression analysis but also a standardized platform for the study of single cells.

Why study single cells?

Because tissues are composed of heterogeneous mixtures of cells, gene expression measurements based on the homogenized population don’t account for the small but critical changes occurring in individual cells.

Single-cell analysis can be critical in applications such as candidate drug screening, cell differentiation and stem cell studies, and measuring individual cell responses to specific stimuli.

The use of a standardized platform such as the Ambion® Single Cell-to-CT™ Kit allows results from single cells to be comparable across the research community. This will accelerate single cell–based applications such as the evaluation of biomarkers from limited clinical samples or other precious samples.

Single-cell Gene Expression Applications

Rare cells or events

  • Circulating metastatic cells
  • Fetal cells in maternal blood
  • Events within a library

Scarce, precious sample

  • Archival tissue
  • Clinical sample (fresh tumor)
  • Biomarker discovery
Single-cell precision in populations

  • Drug candidate screening
  • Cell differentiation (e.g., stem cells)
  • Stochastic responses to stimuli

Answers

1). Can I detect miRNAs 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.

2). Can the 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.

3).  For single-cell analysis, 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.

4).  Do samples need to be frozen immediately?

Samples can be placed at room temperature for up to 30 minutes following lysis, and up to 2 hours after adding Stop Solution. Cell lysates, cDNA, and preamplification samples can all be frozen for future processing.

5).  Any special considerations when evaluating expression of GC rich genes?

No concern for GC rich genes.

6).  Have we used Cells to Ct on plant tissue?

We have not used it on 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 (3) liquid nitrogen-mortar/pestle.

7).  Can we run multiplex reaction with the Cells to Ct kits?

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 Taq Man® Assays.

8).  Is gDNA contamination an issue when using the Cells to Ct™ kits?

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.

9).  Can laser micro-dissected FFPE single cells be used with the Single Cell-to-CT™ Kit?

While we have not tried this in-house, it 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 @ 80°C before adding the RT in order to get good cDNA conversion should improve reverse transcription.

10).  Will the Single Cell-to-CT™ Kit yield enough material for single cell profiling by microarray analysis rather than RT-PCR?

Yes, it will be enough material for microarray analysis. The volume of the sample after the PreAmp step is 26.5 µL which is diluted 1:20 for a total volume of 530 µL, which is more than enough for a large number of TaqMan qPCR assays, TLDA cards or use in the QuantStudioTM open array.

11). What is the recommended method to make single cell equivalent samples?

100 cells are lysed in 10 µL lysis/Dnase I buffer, giving a final lysate conc. of 10 cells/µL.  90 µL of 1X RT buffer is added for a final volume of 100 µL, resulting in a final conc. of this lysate of 1 cell/µL.  Using 1 µL of this lysate will be the equivalent of 1 cell worth of RNA.  It is not making a single cell suspension, however.

12).  How do you prepare single cell suspensions?

We sort cells by making a 1x106 cells/mL suspension in PBS.  These cells can then be stained Live/Dead or conjugated with antibodies specific for various cell type markers and then sorted using the  FASCAria.  The instrument can be set up to dispense a single cell into PCR tubes or 96/384-well plates.

 

Excellent gDNA Removal with Cells-to-CT™ Kit Compared to Competitors

 

10,000 HeLa cells were prepared according to the manufacturer’s instructions and subjected to reverse transcription with (+RT) or without (-RT) reverse transcriptase. PPIA expression levels in each sample were detected by real-time PCR. The resulting +RT CT value was subtracted from the –RT CT value to obtain the ∆CT.

 

Better sensitivity and efficient gDNA removal of Cells-to-CT compared to boiling

 


Eight biological replicates of 10 HeLa cells were placed in 384-well optical plates for each preparation method.  Separate preamplification reactions were performed on –RT reactions for 18S to determine DNA contamination. Cells-to-CT™ reagents are able to remove significantly more gDNA than boiling.