results during qRT-PCR or dPCR, meaning that no genomic amplification occurred during our study (data not shown). To demonstrate that single cell qRT-PCR
resulted in the desired PCR fragments, we sequenced PCR products from individual neurogliaform and pyramidal cells. Although we used validated TaqMan probes in the present study, we sequenced PCR products for all of the amplified genes. Using BLASTN homology searches, we found a 100% match to each gene sequence (data not shown). Tese data confirm that minimal to no amplification of genomic DNA occurred and that amplifi- cations were highly specific.
Strengths and limitations One of the strengths of our PCR protocol
compared to other end-point amplification based PCR methods is that the detection of positive signals is based on individual real-time PCR read-outs. By using the calling algorithm in the OpenArray soſtware and having different thresholds for CT
dence intervals and cycle cutoff values, most of the false positive hits generated by primer dimers or nonspecific amplifications can be excluded. Partitioning the sample into hundreds
or thousands of independent reaction holes (in the case of OpenArray plates) or reaction chambers (using the Fluidigm technology) during dPCR increases the detection sensi- tivity of specific DNA molecules by increasing the target-to-background ratios (12,14,16). Here we described the combination of
whole-cell patch-clamp recording with digital analysis of specific mRNA or miRNA using high-density nanocapillary dPCR. The accuracy and sensitivity of the method was validated with spike-in templates as well as by detecting cDNA from genes with low levels of expression. One of the main advan- tages of this approach is that accurate digital gene expression analyses can be performed on electrophysiologically defined single neurons. Te real-time PCR based dPCR protocol, along with the application of different thresholds for confidence CT
and cycle cutoff values led to
low rates of false positive calls. However, the limitations of the technique are the relative high cost per sample and low-throughput, which restricts the number of genes that can be analyzed from the same cell.
Tis work was supported by the following grants: ERC Advanced Grant, EURYI, NIH N535915 and the Hungarian Academy of Sciences (G.T.), and GOP-1.1.1-11-2011-0003 from the National Investment Agency (NIH) (Avidin, BRC).
Vol. 54 | No. 6 | 2013
Competing interests Te authors declare no competing interests.
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Received 07 January 2013; accepted 24 April 2013.
Address correspondence to László G. Puskás, Avidin Ltd., Szeged, Hungary. E-mail: laszlo@avidinbio- tech.com
or to Gabor Tamás, Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences Department of Physiology, Anatomy and Neuroscience, University of Szeged, Hungary. E-mail: firstname.lastname@example.org
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