In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.
Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8
1. Cell Culture
Our lab uses primary neuronal rat hippocampal cultures for the experiments below. The following describes the modified Banker protocol for how these cell cultures are created and maintained.9 There are, of course, an exhaustive number of permutations of these cell culturing techniques and any established method tailored to the specific needs of a particular laboratory that provides a consistently healthy supply of cells would be a suitable substitute.
2. Preparing Pipettes
Note on RNases: The skin, saliva, and even breath are major sources of RNases, enzymes that degrade RNA. It is imperative that RNase-free technique be observed from this point on in the protocol so that the sample does not become contaminated with RNases and subsequently degrade. This includes always wearing gloves when handling the samples and reagents, never talking over samples or reagents, and using new boxes of sterilized pipette tips and tubes. Often, pipettes that are not designated exclusively for RNA work are decontaminated by wiping them down with an RNase treatment solution such as RNase AWAY (Molecular Bioproducts). However, these solutions will inhibit any downstream enzymatic reactions so it is also important to prevent contamination of your samples with these treatments.
3. Preparing Culture, Tubes, and Microscope
*With our setup it is necessary to use the lid of a 35 mm dish since the walls of the actual dish are too high to allow proper advancement of the micropipette towards the coverslip.
4. Harvesting Cells
5. Saving the Cell
6. aRNA Amplification
7. Representative Results
Successful harvest of a single cell from primary neuronal cultures can be completed in less than 2 minutes, depending on aptitude (see Figure 1). However, time for harvest will vary between systems and with intervening experimental manipulation. Subjecting the single cell to the aRNA procedure (see Figures 4A and 4B) results in microgram amounts of total amplified aRNA and produces a characteristic broad peak when analyzed with a bioanalyzer (see Figure 3). Three rounds can be completed in a minimum of three days, allowing for quick analysis of single cell gene expression.
Figure 1. Shown is an example of a successful harvest of an isolated neuron. We selected a relatively low-density region (A) and advanced the pipette tip toward the desired cell (B). The third image (C) shows the field of view after the cell had been harvested. Note that the surrounding processes remain on the coverslip.
Figure 2. Shown are two images of pipette tips, which are an inappropriate size for effective harvesting. These tips will lead to incomplete harvest (A) and harvest of surrounding mileu (B) respectively.
Figure 3. Following harvest and amplification, analysis using a bioanalyzer is recommended to examine the distribution and quantity of amplified RNA. A successful amplification from single cell material will yield total amounts in the low micrograms and will have a distribution that is smooth and wide.
Figures 4. Schematics of the first round (A) and the second round (B) of the aRNA procedure are shown.
Notes and Troubleshooting
General tips on molecular biological techniques
General Outline of the aRNA Procedure
Figure 4A illustrates the first round of the aRNA procedure. In the first strand reaction, the poly-T portion of the T7-oligo(dT) primer selects for mRNA species (long white rectangle) by binding to the polyA tails. Some microRNAs are also polyadenylated and will be captured by this procedure. More importantly, however, the most abundant RNAs in the cell, ribosomal RNAs, will not. This oligo acts as a primer for Reverse Transcriptase to synthesize a complementary strand of cDNA (long grey rectangle) using the mRNA as a template. The T7 portion of the T7-oligo(dT) primer incorporates the T7 RNA polymerase promoter in frame with the sequence antisense to the starting mRNA. This is used later in the in vitro transcription reaction.
Next, the mRNA in the mRNA/DNA hybrid created in the preceding step is partially hydrolyzed by Rnase H creating RNA “primers” (small white rectangles) similar to the Okazaki fragments created in lagging strand DNA synthesis. DNA Polymerase I uses the RNA fragments to prime DNA synthesis using the DNA complementary to the mRNA as a template. When it reaches the next RNA fragment, its 5′ to 3′ nuclease activity removes the ribonucleotides and replaces them with deoxyribonucleotides. DNA ligase is added to ligate any strands where the replacement of the leading strand is not complete. T4 DNA Polymerase is added to fill in the areas where RNA fragments served as initial primers for DNA Polymerase I creating a blunt-ended double stranded cDNA that is then purified before performing the IVT reaction.
In the IVT reaction, T7 RNA polymerase binds to the T7 promoter incorporated into the double stranded cDNA and synthesizes antisense RNA molecules (long black rectangles) using the sense strand as a template. This serves as the amplification step in which thousands of antisense RNA molecules are produced from each double stranded cDNA molecule (Figure 4A).
The second round, as depicted in Figure 4B, begins with a reverse transcription reaction that is slightly different from that of the first round since the starting RNA is antisense (solid black rectangle) and lacks the polyadenylated tail that was targeted by the T7-oligo(dT) primer in the first round. Therefore, this reaction is primed with random primers (small grey rectangles) and the RNA subsequently denatured. The second strand reaction is then primed by the T7-oligo(dT) primer, which binds to the poly-A sequence at the 3′ end of the sense RNA created in the preceding reverse transcription reaction. Another IVT reaction is performed in the same manner as in the first round. This second round is usually repeated at least once to achieve three rounds of amplification from a single cell.
Applications
The techniques that we have presented in this article can be translated into a large number of applications. The single cell isolation protocol can be modified for use in acute slices.14 Although technically more challenging, the same principles apply in this alternate preparation. Additionally, if the size of the pipette is slightly adjusted, recordings of the physiology of the cells can be made before harvest allowing for a well-controlled investigation of molecular mechanisms behind physiological outputs. Another slight modification is to isolate processes from the cell soma.15 For this application, collect cell bodies with one pipette and then go back with a fresh pipette and collect 100-300 identified dendrites or axons per collection tube.16
Once cells have been harvested, comparisons of mRNA abundances and compositions can be made between different and even within the same cell populations. Incorporating biotinylated-UTP into the third round aRNA allows for microarray analysis to determine these relative mRNA abundances. The composition of the original mRNA population can also be determined after the aRNA procedure using next generation sequencing. The amplified aRNA can also be used to confirm cell phenotype conversion studies in which a full set of mRNAs from one cell type are transfected into a different cell type in order to induce transition of the phenotype of the latter cell type into that of the former, a procedure developed by the lab and known as TIPeR.17 These studies are particularly useful for studying disease states and cell phenotypes and such studies are currently ongoing in the lab. RT-PCR or qPCR can be performed on the amplified material to confirm the expression of cell-specific genes. Additionally, evaluations of the efficiency of transfection or transduction can be made at the single cell level.
Advantages and Limitations
As stated in the abstract, isolation of single cells for analysis eliminates the averaging effects seen with analysis of heterogeneous cell populations. These averaging effects misrepresent mRNA abundances within a single cell by over-representing abundant transcripts and averaging out and preventing detection of many low-abundance transcripts. Flow cytometry can be used to sort individual cells, but this method requires knowledge of cell specific markers and expensive equipment.18 Laser capture microdissection either with UV or IR laser capture microdissection systems allow for single cell and even subcellular capture but requires cells of interest to be located at the surface of very thin sections.19 Similar to flow cytometry, laser capture microdissection also requires expensive equipment.
One of the major benefits of the electrode based collection technique described above is that valuable electrophysiological data can be obtained from the cell of interest before harvest, allowing for functional and transcriptome analysis to be performed on the same cell.20 A downside of our technique is that it does require experience using micromanipulators. Investigators familiar with micromanipulators will find this technique very intuitive; however, individuals with no such experience will need to become comfortable with the fine movements required.
Inherent in any amplification technique is the preferential amplification of certain transcripts based on size and nucleotide composition.4,6,7 Polymerase chain reaction (PCR) based techniques such as reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) result in exponential amplification of transcripts, whereas the aRNA amplification procedure results in linear amplification. Thus, one of the major advantages of the aRNA procedure lies in its ability to better preserve the relative abundances of mRNA transcripts by only linearly amplifying any errors or biases that occur in the amplification process as opposed to exponentially amplifying said errors and biases.
The aRNA procedure coupled with microarray analysis allows for comparison of mRNA abundances between single cells of similar or different morphology, treated or untreated. Additionally, amplified material can be submitted for next generation sequencing.5,8 However, care should be taken in such analyses since, in contrast to the use of random primers for the procedure, the oligo-dT primed procedure described above biases amplification of the 3′ ends of mRNA and generally results in slight shortening of subsequent amplified material with each round. Care should be taken in analyzing microarray results with standard methods as some false absent calls can arise from slightly shortened amplified material. In addition, while sequencing results will indeed provide the full 5′ sequence of most of the original mRNA, the 5′ sequences of some mRNA might be missed. For these reasons, the number of rounds of amplifications should be limited.
The authors have nothing to disclose.
Thank you to Kevin Miyashiro for plating and maintaining cell cultures, to Dr. Terri Schochet for providing cell cultures for the pictures included in this document. In addition, thank you to Kevin Miyashiro, Dr. Peter Buckley, and Tiina Pertiz for input on the aRNA procedure. Funding for this work was from the National Institute on Aging, the National Institute on Mental Health and the Human Resources Fact Finder funds from the Commonwealth of Pennsylvania.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Spiegelgas coverslips | Carolina Biological | 63-3029 | ||
Nunc 35×10 mm culture dishes | Fisher | 12-565-90 | ||
Water for cell culture | Lonza | 17-724Q | For making solutions used for cell culture and rinsing coverslips | |
Poly-D-Lysine MW70-150K | Sigma | 6407 | ||
Laminin, ultrapure | BD Bioscience | 354239 | ||
Boric acid | Sigma | B0252 | ||
MEM with Earle’s salts and glutamax | Invitrogen | 41090-101 | For plating cells | |
D-glucose | Sigma | G8769 | ||
Penicillin-streptomycin | Invitrogen | 15140-122 | ||
Horse serum | Invitrogen | 16050 | ||
Cytosine beta-D-arabinofuranoside | Sigma | C1768 | ||
MEM with Earle’s salts and L-glutamine | Invitrogen | 11095-098 | For growing cells | |
Sodium pyruvate | Sigma | P2256 | ||
B27 serum-free supplement | Invitrogen | 17504-044 | ||
Borosilicate glass capillary tubes (1.5mm O.D, 100mm length) | Kimble Chase | 34500 99 | Any borosilicate glass pipette will work as long as the proper bore size is attained. | |
HBSS | Invitrogen | 14175 | Any solution will work as long as the components won’t interfere with any future processing (i.e. no Ca2+ or Mg2+) | |
1ml syringe | BD | 309628 | ||
Needle | BD | Gauge depends on the diameter of the pipettes | ||
dNTP mix | Amersham | 28-4065-51 | ||
dt-T7 oligo | Custom | Midland Certified | ||
Second strand buffer (5x) | Invitrogen | Y01129 | ||
DTT | Supplied with second strand buffer | |||
E.coli DNA Ligase | Invitrogen | 100002324 | ||
DNA polymerase I | Invitrogen | 100004926 | ||
Rnase H | Invitrogen | 18021-071 | ||
Megascript T7 kit | Ambion | AM1334 | ||
Random primers | BMB | 11034731001 | ||
Superscript III Reverse Transcriptase | Invitrogen | 56575 | in kit (18080-044) comes with first strand buffer (Y02321) and DTT | |
MEGAclear Kit | Ambion | 1908 | ||
MinElute Reaction Cleanup Kit | Qiagen | 28206 | ||
T4 DNA polymerase | Invitrogen | 100004994 | ||
Rnasin | Promega | N251B | ||
Illumina TotalPrep RNA Amplification Kit | Ambion | AMIL1791 | ||
Flaming/Brown micropipette puller | Sutter Instruments | P-87 | Sutter has many other models, many are discussed in the cookbook | |
Micromanipulator | Olympus | Many other micromanipulators will work such as the newer Eppendorf models | ||
Pipette Holder | Warner Instruments | MP-S15A | Will vary with micromanipulator and pipette O.D. | |
Bioanalyzer RNA 6000 Nano Kit | Agilent | 5067-1511 |