Richard Fekete, Ph.D. Discusses Single Cell Analysis in an Insightful Webinar
by Jennifer Hornstein - 04/13/12
A free single cell analysis webinar is now available. Listen to a discussion about analyzing expression profiles from single stem cells with Richard Fekete, Ph.D. of Life Technologies.
What is single cell analysis and why study it?
We all know that tissues are composed of heterogeneous mixtures of cells. But what does that mean when we take gene expression measurements from homogenized samples? Are we accurately accounting 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.
About The Ambion® Single Cell-to-CT™ Kit
Pre-optimized protocol - The Ambion® Single Cell-to-CT™ Kit presents a complete validated workflow for gene expression analysis for samples containing between 1-10 cells.
Materials - This single cell analysis kit contains reagents for sample preparation, reverse transcription, pre-amplification and qPCR that have been optimized together.
Workflow - Each workflow begins with a simple 7 minute sample prep where cells are lysed into a solution that is compatible with downstream RT-PCR - no purification needed.
Sensitivity - This is followed by VILO™ with Superscript for reverse transcription, the TaqMan® PreAmp Master Mix to amplify the cDNA, and TaqMan® Gene Expression Master Mix for qPCR with 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.
Common questions from customers:
A series of frequently asked questions prompted this webinar. Listen in as Richard covers the questions below and more.
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). 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.
7). 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.
Watch the single cell analysis webinar now.