1Department of Structural and Molecular Neuroscience, McLean Hospital, 2Department of Psychiatry, Harvard Medical School, 3Department of Psychiatry, Beth Israel Deaconess Medical Center
Pietersen, C. Y., Lim, M. P., Woo, T. W. Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue. J. Vis. Exp. (30), e1444, doi:10.3791/1444 (2009).
We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51μg, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.
1) Tissue sectioning:
We obtained the necessary tissue (control n=9, schizophrenia n=9) from the Harvard Brain Tissue Resource Center, matched for age, sex and postmortem interval (PMI) (Table 1). Liquid nitrogen vapor blocks were approximately 3mm thick were taken from Brodmann's area 42 (superior temporal gyrus).
Before beginning staining of pyramidal neurons, conditions must be optimized in order to obtain optimal tissue "lift" and high RNA quality. Tissue "lift" describes the process during laser-capture microdissection, where the neurons marked for capture adhere to the cap and are separated while the remaining tissue is left behind.
We have previously determined that for pyramidal neurons, 500 cells per case are adequate in order to acquire enough RNA for microarray hybridization. Approximately four sections are needed per case, mainly due to sectioning and staining artifacts.
2) Staining of pyramidal neurons:
Identification of pyramidal neurons is achieved with the Histogene quick staining solution and kit, as it allows us to visualize these neurons, without prolonged exposure to aqueous solutions, thereby preserving RNA.
The staining steps should only be performed if laser-capturing immediately thereafter. All solutions should be changed every 4-6 slides in order to prevent contamination and to ensure proper dehydration.
3) Single cell laser-capture microdissection:
After staining, pyramidal neurons from layer III are removed using laser-capture microdissection, with the Arcturus XT system and software. Briefly, the system works by pulsing an adjustable strength IR laser through a thermoplastic film located at the base of a cap, resulting in the extension/distension of the film directly onto the cells of interest. When the cap is lifted from the tissue, the cells remain captured/captive on the cap. A pictorial summary is given in Figure 1.
Before starting this procedure, it is important to distinguish between the two types of caps available with this system. While the Macro CapSure cap is generally used for capturing larger tissues structures, the CapSure HS caps are designed to capture small numbers of individual cells and therefore these caps have a smaller surface area designated for capture. The HS caps also have rails that prevent the cap from being in direct contact with the tissue section, while the Macro cap does not. These rails reduce the effect of tissue folding that may affect or induce variability of spot size (of the laser pulse) as the Macro cap sits directly on folded, uneven tissue. For our procedure, we used the HS caps to take advantage of their ability to reduce the effect of tissue folding, but kept the Macro cap settings on the software in order to maximize the captured area as we are capturing a large number of cells.
We now adjust the power and duration of the laser pulse in order to capture the pyramidal neuron. This is particularly important, as it determines the specificity of the cell captured in addition to the number of cells captured. If the pulse is too weak not all identified neurons will be captured, while if the pulse is too strong it could capture surrounding tissue/cells other than the neuron alone. Generally, with the tissue and cell specifications employed here, the laser strength (70mW) and duration (16msec) result in a spot size of 25μm, approximately in line with pyramidal neuron size (Fig. 1B). However, these settings are fine-tuned to each section in order to obtain roughly the same spot size per case. If "lift" is compromised, one can always increase the number of cells captured in order to keep the amount of RNA obtained from each case comparable. However, be sure to keep your capture time including staining under 1.5 hours, as longer than this can compromise RNA quality.
4) Obtaining RNA from specific neuronal populations:
One can go directly from capturing to isolation, or thaw the sample stored in the -80°C freezer. In our experiment, we first collected all the samples needed and stored them at -80°C before proceeding to RNA isolation.
RNA isolation is performed using the PicoPure Isolation Kit, which is designed to isolate small numbers of cells from LCM samples and to retain low-abundance mRNA. Generally, we follow the protocol that is included with the kit. Below, we give a basic description of the process.
Quality control is very important when isolating RNA, especially when the quantity is small and the amount needed, such as for microarray experiments (15μg), is large. The extracted RNA therefore must undergo quality control at various time points throughout the process. The first occurs after extraction. We establish the total RNA integrity via gross examination of the 18S/28S peaks of the electropherograms and virtual gel generated by Experion HighSens LabChip. This method is quick and provides two means of measurements to check RNA quality- an electropherogram and a virtual gel (Fig. 2). If the quality of the total RNA is relatively good (Fig. 2A, B vs. C, D), we then proceed with linearly amplifying the mRNA, which accounts for roughly 2% of the total RNA.
5) Linear amplification:
Amplification of RNA extracted from laser-captured tissues is necessary in order to produce an adequate amount of RNA for subsequent microarray hybridization and qRT-PCR experiments. In order to minimize the possibility of potential confounds noted with T7-based linear amplification, we minimized the number of rounds of amplification necessary by maximizing the amount of the starting laser-captured tissue materials.
Our preliminary data (not shown) suggest that two rounds of linear amplification (RiboAmp HSPLUS kit) of total RNA extracted from tissues that contain approximately 500 cells would produce approximately 50μg of aRNA -- sufficient for the performance of both microarray and qRT-PCR experiments. If possible, avoid unnecessary transferring of sample between 0.2ml and 0.5ml tubes by using 0.5ml block in the thermal cycler and the 0.5ml tubes provided in kit.
Again, the protocol followed is the one provided with the kit. An abbreviated version is described here.
The RNA undergoes another round of linear amplification (with separate primers - see kit protocol), after which more quality control steps are employed. One ul of the sample is diluted to approximately 250ng/ul to determine the mRNA transcript length and concentration with the StdSens LabChip (Experion). Traditional Affymetrix microarray technology requires the mRNA to be at least 600 nucleotides in length in order to be detected. The gel and electropherogram obtained from the LabChip should therefore depict this minimum mRNA transcript length (Fig. 3).
An additional quality control is determined via a NanoDrop spectrophotometer analysis, where a ratio made up of two optical densities, i.e. A260 and A280, determines RNA purity. Samples with an optical density around 2.1 should be labeled for gene expression analysis, although ratios up to 2.7 have been shown to have comparable hybridization quality. The concentration should also be between 800ng/μl-1μg/μl, as lower than this has led to RNA degradation while in storage (even at temperatures below -80°C) and has also led to poor microarray results.
6) Biotin-labeling mRNA samples for Affymetrix microarray analysis:
The TURBO Biotin labeling kit (end-labeling) from Molecular Devices is used to label the aRNA obtained from amplified samples. A shortened version of the protocol is given here.
aRNA was then hybridized and gene expression profiled, using the Human X3P GeneChip probe array from Affymetrix. As this chip was designed for more degraded aRNA, we used it as a precaution, as many factors we cannot control can affect the quality of postmortem tissue. As our lab did not have the adequate facilities for hybridization, the process was performed at the Partners Genomic Core facility.
1+2+3) Tissue sectioning, staining of pyramidal neurons and laser-capture microdissection:
The protocol described above should result in two slides with four sections per slide. Each section should be smooth with minimal tearing, cracking and folding. With correct staining, the stain will identify darkly stained pyramidal neurons around 20 - 25μm in size with a pyramidal shape and visible apical dendrites. With the CapSure HS caps at Macro settings, and correct adjustment of the laser power and strength for each section, you should obtain around 500 - 700 cells per section/case resulting in at least 500pg of total RNA. Approximately 85 - 100% of the neurons will adhere to the cap (Fig. 1).
4) Obtaining RNA from specific neuronal populations:
As described, after total RNA isolation, we check the RNA quality by means of an electropherogram and virtual gel via the Bio-Rad Experion. In the electropherogram, you should see two distinct peaks corresponding to the 18S and 28S ribosomal RNA units. With postmortem tissue, however, this is not always the case, as the tissue may be degraded due to factors prior to sectioning. You will normally see a large bump indicating degradation, with a large peak around 18S, and a smaller 28S peak (Fig. 2A, B). As long as the electropherogram profiles are comparable across samples, we have found this guideline to result in mRNA quality good enough to perform both RT-PCR and microarray studies (Imbeaud et al., 2005).
If there is too much degradation - as indicated by a large area under the curve of the electropherogram, or if peaks are not visible (Fig. 2C, D) - the cells should be captured again from the tissue. We would also recommend doing a tissue-scrape test, where RNA is extracted from whole fragments of the section (not just cells), before recapturing the cells to determine whether the tissue block itself is degraded.
5) Linear amplification:
To test the quality of the mRNA after two rounds of linear amplification, we made use of both the Bio-Rad Experion StdSens LabChip and the NanoDrop spectrophotometer. Traditional Affymetrix microarray technology requires the mRNA to be at least 600 nucleotides in length in order to be detected, which was obtained with this protocol. In fact, mRNA lengths were detected into the 1000-nucleotide range. The electropherogram also reflects this with large peaks slowly descending as a function of time (Fig. 3A, B).
The NanoDrop readings indicated an A260/A280 ratio average of 2.5 across samples, indicating viable mRNA (Table 2).
Our results indicate that with this protocol, both the quantity and quality of RNA obtained is good enough to interrogate gene expression differences via the Affymetrix human X3P GeneChip.
6) Biotin-labeling mRNA samples for Affymetrix microarray analysis:
After hybridization to the Affymetrix human X3P GeneChip, we achieved percent calls of an average of 26.6% (Table 2), indicating adequate hybridization and probe intensities.
Table 1: Cohort summary
Abbreviations: F - female, M - Male, PMI - postmortem interval, schizoph. - schizophrenia.
Table 2: Summary of results depicting RNA concentration, quality and hybridization efficiency
|Samples||[mRNA] ng/ul||A260/A280||Probe Intensity||Percent call|
Figure 1: Summary of the laser-capture microdissection process. A) Pyramidal neurons are identified morphologically. B) After the laser has pulsed through the thermoplastic film, the cells adhere to the cap and are therefore no longer on the slide, leaving surrounding tissue behind. C) Photomicrograph of a homogeneous cell population adhering onto the CapSure Cap after laser capturing.
Figure 2: Total RNA quality control. A+B) Represents good quality total RNA after isolation, while C+D) illustrates what total RNA should not look like after isolation. A) An electropherogram with clear 18/28S peaks corresponding with the virtual gel in (B). C) An electropherogram showing a large area under the curve and no 18S/28S peaks indicating RNA degradation. This is also shown in the virtual gel (D).
Figure 3: mRNA quality control. A+B) Electropherogram (A) and virtual gel (B) indicating the mRNA transcript length (spread) necessary for microarray studies, i.e extending past 600 nucleotides (nt). C+D) Electropherogram (C) and virtual gel (D) showing what an insufficient mRNA spread would look like, as the mRNA transcript length does not extend past 200nt.
Here, we attempt to customize a protocol to extract homogeneous cell populations from heterogeneous postmortem brain tissue using the Arcturus XT system and related RNA extraction kits. As outlined above, we have made some modifications to the original protocols provided by the company, in order to maximize RNA integrity. The successful capturing of single cells depends solely on optimizing the steps prior to capture in order to achieve maximum cells with good RNA. These steps include the various parameters associated with tissue sectioning and staining. In short, with regards to tissue preparation these include: 1) decreasing the temperature gradient when sectioning, 2) only placing 2 sections per slide, 3) performing all dehydration steps on ice, 4) adding RNase Inhibitor to the stain, 5) increasing the dehydration duration in the final 100% ethanol to 3 minutes.
With regards to capturing, we feel that the combination of CapSure HS caps, with the software on Macro setting, should ensure optimal results. A humidity-controlled environment is critical to capturing single cells, as high humidity drastically reduces tissue "lift". High temperatures can also have a negative effect on RNA quality.
During the RNA isolation protocol with the PicoPure kit, there are two wash steps. It is important to check that all wash buffer is removed before transferring to another microcentrifuge tube for RNA elution, as any presence of the buffer in your final sample will result in less starting RNA for amplification. To ensure this, we centrifuge the final wash step for 2.5 minutes instead of 2 minutes as suggested in the protocol provided.
For the most part, the linear amplification protocol was according to the manufacturer's instructions. However, two crucial points should be taken into account when using this protocol: 1) try to limit loss of RNA via excess transferring of samples between tubes, by using only the 0.5ml tubes provided in conjunction with a 0.5ml block in the thermal cycler; and 2) when transferring the sample to the purification column for purification of cDNA, be sure to spin down the sample tubes after the first transfer. This will result in an additional 1-2ul of sample to add to the purification column and therefore can increase cDNA recovery.
When making use of the TURBO end-labeling kit for biotin-labeling of your limited aRNA samples, we suggest adding an extra few μl's of water before the centrifugation step, to increase the labeled RNA yield extracted from the column. An extra minute of centrifugation will also help in this regard, especially if you are working with degraded tissue, such as FFPE tissue.
We feel confident that this protocol will enable the user to isolate small homogeneous structures, such as single cell populations, within heterogenous postmortem brain tissue. This reduces dilution effects of surrounding structures and other cell-types located in the various layers within the brain, leading to results targeted towards the specific cell types under investigation. As the quality of the resulting RNA is good enough for microarray studies, we can begin to pinpoint specific molecular signatures of specific cell populations affected in disease states.
The production of this video-article was sponsored by Life Technologies.
We gratefully acknowledge Molecular Devices Analytical technologies/Arcturus for their generous donation of reagents used for the study. We also thank the Harvard Tissue Resource Center for providing postmortem human brain tissue. Funding: RO1MH76060 and P50MH080272.
|4.5€¯ Forceps||VWR international||82027-392|
|Arcturus XT™ Laser-Capture Microdissection (LCM) System||MDS Analytical Technologies||13821-00|
|CapSure™ HS LCM Caps||MDS Analytical Technologies||LCM 0214|
|CapSure® Macro LCM Caps||MDS Analytical Technologies||LCM 0211|
|Experion™ Automated Electrophoresis Station||Bio-Rad||700-7001|
|Experion™ RNA HighSens Chips||Bio-Rad||700-7155||10 chips|
|Experion™ RNA HighSens Reagents||Bio-Rad||700-7156||10 chips|
|Experion™ RNA StdSens Chips||Bio-Rad||700-7153||10 chips|
|Experion™ RNA StdSens Reagents||Bio-Rad||700-7154||10 chips|
|Falcon® polypropylene Conical tube||BD Biosciences||352070|
|GeneAmp® thin-walled reaction tube with domed cap||Applied Biosystems||N8010611|
|GeneChip® Human X3P array||Affymetrix||900516||6 chips|
|Histogene® LCM Frozen Section Staining kit||MDS Analytical Technologies||KIT0401||For staining solution, jars, ethanols and Xylene|
|Histogene™ LCM slides||MDS Analytical Technologies||12231-00|
|Micro Slide Box||VWR international||48444-004|
|Microm HM 505E cryostat||Thermo Fisher Scientific, Inc.||22-050-350||Catalogue number for similar product as current product no longer available|
|Microprocessor Controlled 280 series Water bath||Thermo Fisher Scientific, Inc.||51221048||Model 282|
|Molecular Sieve||EMD Millipore||MX1583L-1|
|NanoDrop 1000 Spectrophotometer||Thermo Fisher Scientific, Inc.||n/a||The NanoDrop 1000 is not available anymore, but the NanoDrop 2000 is comparable.|
|PEN Membrane glass slides||MDS Analytical Technologies||LCM 0522|
|PicoPure® RNA Isolation kit||MDS Analytical Technologies||KIT0204|
|RiboAmp®HSPLUS with Biotin labeling||MDS Analytical Technologies||KIT0511B||12 reactions Includes TURBO biotin labeling„¢ kit|
|RNase-Free DNase Set||Qiagen||79254|
|RNasin® Plus||Promega Corp.||N261B|
|SuperScript™ III Reverse Transcriptase||Invitrogen||18080-044|