Reports


responses to current pulses as well as their axonal and dendritic morphology (Figure 2) (20–24). To demonstrate the utility, reproducibility,


and sensitivity of our protocol, the expression of the housekeeping gene rps18 and a potential cell-type dependent marker gene, gamma- aminobutyric acid (GABA) A receptor delta subunit (gabrd) were recorded by dPCR from individual cellular aspirates from the three different neuron types. Aſter recording the firing pattern for each neuron (22), the cellular content was aspirated and used for cDNA synthesis. Five independent cells from each type were collected from three brain slices in separate experiments and measurements of transcript numbers for each gene were repeated five times in an independent assay. Transcript numbers for each cell are listed in Table 1. Te expression of rps18 was found to be


similar in each type of neuron (59–67 copies/ cell), with low CV values (30%–40%) (Table 1). On average, only two gabrd mRNA copies could be detected in pyramidal cells (with a standard deviation of 1.22); a few transcripts were recorded in fast-spiking neurons (7.60 ± 4.28), representing gabrd, a specific gene with low expression levels, while over 30 messages could be recorded in neurogliaform cells (Table 1).


Cell-type specific miRNA expression analysis of single neurons with dPCR To understand the role of miRNAs in different neuronal processes, including pathological alter- ations, there must be an understanding of how different miRNAs are expressed in different cell types. Multiplex miRNA expression profiles were determined in single cells with qRT-PCR (25). Recently, the applicability of the high-density OpenArray protocol has been demonstrated for the detection of miRNA expression profile by stem-looped RT-PCR (26). As a model, we used the OpenArray plates for dPCR studies of a specific miRNA, mir-132, from cytoplasm harvested into patch pipettes from single neurons of different types. Specific miRNA was converted into cDNA using specific stem- looped primers. The total RT reaction was amplified in 256 individual reaction holes on the array as described above. As an example, the number of mir-132 molecules was determined in pyramidal cells, fast spiking cells, and neuro- gliaform interneurons. Dysregulation of mir-132 has been found in schizophrenia and mir-132 is also involved in neurodevelopmental processes; however, there are only limited data on cell-type specific expression (27). By using our dPCR protocol, mir-132 expression was found to be similar in the three cell types (Table 1, Figure 3), although differential expression cannot be excluded in these cells during different periods of neurodevelopment or under pathophysiological conditions.


Vol. 54 | No. 6 | 2013 In order to demonstrate the sensitivity of


our protocol, a miRNA with low expression levels was selected for analysis in neurons. In our preliminary single cell qRT-PCR studies, we found that the expression of mir-184 was very low or even undetectable by standard protocols in both neurogliaform and pyramidal cells (data not shown). By using our combined patch-clamp and dPCR approach, we detected an average of 2.33 ± 1.58 (n = 3) copies in pyramidal cells and 3.33 ± 1.52 (n = 3) copies in neurogliaform cells, while RT negative controls resulted in 0.33 ± 0.58 (n = 3) false positive calls.


Demonstration of specific mRNA and miRNA expression in single cells with traditional qRT-PCR To confirm mRNA (rps18 and gabrd) and miRNA (mir-132) expression in single neurons analyzed by our dPCR approach, classical single cell qRT-PCR was run on all three neuron types used in this study (Figure 3). Analysis of ∆CT


values obtained


by classical single-cell qRT-PCR produced results corresponding to those obtained with dPCR for both mRNA and miRNA (Table 1). Expression of gabrd was specific to neuro- gliaform cells (average CT


value of 23.32), with


significantly lower expression levels detected in fast spiking cells. No amplification was recorded in pyramidal cells. CV values were determined for each neuron


and each gene analyzed. We found that our dPCR protocol resulted in data with small CV (8%–60%), while CV values obtained from traditional PCR were high (187%–2025%), likely because of low quantities of input DNA in the reactions. From these data, we conclude that dPCR in combination with single cell patch-clamp technology enables absolute quantitation of transcript copy numbers in a reproducible manner.


Physiological alteration of gene expression in single neurons determined with dPCR The approach described here can also be used to isolate and evaluate single abnormal neurons in various disease models of the central nervous system (CNS). In a model experiment, oxidative stress was introduced by applying hydrogen peroxide at a 0.3 mM final concentration onto cortical slice prepa- rations submerged in artificial cerebro- spinal fluid for 2 h. Pyramidal cells were harvested by patch-clamp from non-treated


Table 2. mRNA average copy numbers of untreated and H2 dPCR


PC rps18 hmox1 hspb1


60.25 (21.04)


18.00 (3.00)


24.00 (4.36)


334


63.33 (11.37)


29.25 (3.59)


38.5 (6.45)


O2


Figure 4. Oxidative-stress dependent expression changes. mRNA copy number changes of hmox1 and hspb1 in pyramidal cells. Results of no tem- plate controls for all the genes analyzed with dPCR (negc) are also shown.


and hydrogen peroxide treated brain slices. dPCR of the samples was run on OpenArray plates to determine the relative mRNA copy number changes of hmox1 and hspb1 genes. Both genes have been implicated in oxidative stress and methamphetamine-induced neuro- toxicity (28). We found that this short period of oxidative stress did not alter the number of the house-keeping rps18 transcripts (P = 0.918), but significantly elevated the number of mRNA molecules of both hmox1 and hspb1 genes (P < 0.05; Table 2, Figure 4).


Demonstration of specific amplification during PCR To demonstrate the specificity of TaqMan probes used in single cell qRT-PCR as well as in dPCR, control parameters were tested including a reverse transcription negative control (where all the reagents except the RT reaction mix were applied), intronic template DNA, and intergenic template DNA. Tese reactions resulted in no amplification with qRT-PCR and few or no amplification products (maximum of 2) with dPCR (Table 1).


To determine the background noise for


gabrd, we repeated the amplification of the negative control reaction 7 times and found an average of 0.43 signals with a standard deviation of 0.53. To eliminate the possibility of amplifying


genomic DNA, we tried to amplify multiple introns of the ins2 gene as well as an intergenic region at the ifngr1 gene locus on chromosome 1. None of these primer sets gave positive


-treated pyramidal cells (PC). Pc + H2O2 No template


0.63 (1.06)


0.50 0.71)


0.00 (0.00)


n 7 4 4


P value 0.918


0.007 0.02


www.BioTechniques.com


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68