The following single cell applicable techniques are being used at Penn, either in core facilities or in individual labs. This enumeration of these techniques is intended to provide a foundation from which an investigator can identify potential methodologies (and experts at using them) that might be useful for their research efforts.
Single Cell Isolation Methods
PATCH PIPETTE ISOLATION
A patch-clamp pipette can be used to isolate the contents of a single cell, either from culture or from whole tissue (e.g. brain slice).
- Morris J, Singh JM, Eberwine JH. Transcriptome analysis of single cells. J Vis Exp. 2011;25:2634.
ISORAFT ISOLATION
The Isoraft system, developed by Cell Microsystems, is an alternative method for isolating single cells from culture. Cells are cultured on an array of magnetic micro-rafts. A simple microscope and release device are used to dislodge and capture individual rafts containing cells of interest.
- Cell Microsystems: http://www.cellmicrosystems.com/products/release-device/
- Isoraft video demonstration: http://www.youtube.com/watch?v=M8cUMzFf8uk&list=PLui-9CCDVuXawnp6ml3K0al1D2vXkSz4c
C1 SINGLE CELL AUTO-PREP SYSTEM
The C1 System from Fluidigm provides an automated, microfluidics based platform for isolation of some dispersed single cells and RNA amplification by PCR, Nugen Ovation and aRNA. A C1 system is available in the Next Generation Sequencing Core.
- Fluidigm: http://www.fluidigm.com
- Workflow video: http://www.fluidigm.com/c1-system-workflow-video.html
RNA Amplification Methods
LINEAR RNA AMPLIFICATION (aRNA)
mRNA from a single cell can be amplified using an oligo(dT) primer with a T7 promoter sequence, in conjunction with T7 RNA polymerase. Unlike PCR-based amplification, this method allows for linear amplification of the RNA, preserving the original expression level ratios between different mRNAs. Two or more rounds of linear amplification yields sufficient material to perform RNA-seq on a single cell.
- Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD, Eberwine JH. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A. 1990;87:1663-7.
- Eberwine J, Yeh H, Miyashiro K, Cao Y, Nair S, Finnell R, Zettel M, Coleman P. Analysis of gene expression in single live neurons. Proc Natl Acad Sci U S A. 1992;89:3010-4.
- Morris J, Singh JM, Eberwine JH. Transcriptome analysis of single cells. J Vis Exp. 2011;25:2634.
SMART-SEQ RNA AMPLIFICATION
Smart-Seq is a PCR-based method for RNA amplification. It takes advantage of the properties of MMLV reverse transcriptase to add adapter sequences to both the 5’ and 3’ ends of synthesized cDNA, allowing for PCR amplification. It is less accurate than aRNA, but is quicker and simpler to perform.
- Ramsköld D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R.Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol. 2012;8:777-82.
RNA-seq
RNA-seq uses next generation sequencing technology to sequence and identify every mRNA species in a sample. It improves upon previous microarray technologies because it does not rely on previous knowledge of the transcriptome (i.e. novel transcripts can be identified), has a greater dynamic range and is more accurate.
Next generation sequencing is available through the Next-Generation Sequencing Core, the Penn Genomic Analysis Core and the High-Throughput Sequencing Center at CHOP.
One of the most commonly used next generation sequencing technology is the Illumina platform. More information about next generation sequencing and Illumina sequencing can be found here and here.
PPSCB is happy to provide RNA-seq datasets, generated by participants in our National Single Cell Transcriptomics Workshops, for public use. The datasets and associated protocols are available for download here.
Mass Cytometry
The CyTOF mass cytometry platform combines flow cytometry with mass spectrometry to allow analysis of a large number of protein markers in single cells. Cells are stained with a panel of metal-tagged antibodies and then individually analyzed by time-of-flight mass spectrometry. Currently up to 34 different proteins can be simultaneously detected and quantified. A CyTOF instrument was recently purchased jointly by the Philadelphia VA Medical Center and the Penn Institute for Immunology. For more information email ifiadmin@mail.med.upenn.edu.
Other Specialized Technologies
TIVA
Transcriptome in vivo analysis (TIVA) is a novel technology developed in the Eberwine and Dmochowski labs, which allows for photo-activated capture of mRNA from a single cell or sub-cellular region. The TIVA tag is a cell-penetrating, caged molecule that, on activation with a laser, becomes uncaged and anneals to mRNA in the region of interest. The captured mRNA can then be purified from lysed cells by means of a biotin moiety incorporated into the TIVA tag.
- Lovatt D, Ruble BK, Lee J, Dueck H, Kim TK, Fisher S, Francis C, Spaethling JM, Wolf JA, Grady MS, Ulyanova AV, Yeldell SB, Griepenburg JC, Buckley PT, Kim J, Sul JY, Dmochowski IJ, Eberwine J. Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue. Nat Methods. 2014 11:190-6.
TIPeR
Transcriptome transfer (TIPeR) is a method for converting one type of cell into another, by transfection with RNA reflecting the target cell type transcriptome. Research in the Eberwine and Kim labs has shown that transferring mRNA isolated from astrocytes into neurons effects their transformation into astrocytes. Using the same technique, astrocytes and fibroblasts could be converted into cardiomyocytes.
- Sul JY, Wu CW, Zeng F, Jochems J, Lee MT, Kim TK, Peritz T, Buckley P, Cappelleri DJ, Maronski M, Kim M, Kumar V, Meaney D, Kim J, Eberwine J. Transcriptome transfer produces a predictable cellular phenotype. Proc Natl Acad Sci U S A. 2009;106:7624-9.
- Kim TK, Sul JY, Peternko NB, Lee JH, Lee M, Patel VV, Kim J, Eberwine JH. Transcriptome transfer provides a model for understanding the phenotype of cardiomyocytes. Proc Natl Acad Sci U S A. 2011;108:11918-23.
SINGLE MOLECULE RNA FISH
Fluorescent in situ hybridization targeting RNA molecules (RNA FISH) is a technique for visualizing RNA within cells. The quantification of RNA at single molecule resolution using RNA FISH has been pioneered by Dr. Arjun Raj of Penn Bioengineering. The technique relies on the use of multiple fluorescent probes targeted to the same RNA molecule, resulting in superior specificity and signal-to-noise ratio, compared to single probe approaches. Fixed cells and tissues, as well as live cells, can be analyzed.
- Introduction to single molecule RNA FISH: https://sites.google.com/site/singlemoleculernafish/home
