Image_3_A PCR-Based Method for RNA Probes and Applications in Neuroscience.TIF

<p>In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-δ) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.</p

Image_1_A PCR-Based Method for RNA Probes and Applications in Neuroscience.TIF

<p>In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-δ) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.</p

Image_4_A PCR-Based Method for RNA Probes and Applications in Neuroscience.TIF

<p>In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-δ) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.</p

Functional characterization of NBCn2 variants.

<p>The oocytes were superfused with nominally HCO₃⁻-free ND96. An acid load was introduced by exposing the cells to 1.5% CO₂/10 mM HCO₃⁻ which was followed by a removal of extracellular Na⁺. Intracellular pH and membrane potential <i>Vm</i> of the oocytes were simultaneously recorded with microelectrodes. Representative recordings of pH_i and <i>Vm</i> are shown for rNBCn2-C-EGFP (<b>A</b>), rNBCn2-G-EGFP (<b>B</b>), rNBCn2-GΔNt-EGFP (<b>C</b>), and H₂O-injected control oocyte (<b>D</b>). (<b>E</b>) Summary of pH_i recovery rates (dpH_i/dt) of oocytes expressing NBCn2 variants/mutant or injected with H₂O. The dpH_i/dt of NBCn2-expressing oocytes are all significantly different from that of H₂O-injected control oocytes.</p

cDNAs of <i>SLC4A10</i> orthologs from different species.

<p>Note: A series of ESTs and cDNAs from the brain of human, mouse, and rat that encode the Nt of MEIK-NBCn2 are identified in GenBank. The accession numbers of these sequences are not listed here since the expression of MEIK-NBCn2 has been well studied in these species.</p

Characterization of mouse <i>Slc4a10</i> promoters.

<p>(<b>A</b>) Diagram of the minimal promoter regions of mouse <i>Slc4a10</i>. <i>Slc4a10</i> contains two alternative promoters: the distal P1 and the proximal P2. Shown here are only the first three exons (E1–E3) of <i>Slc4a10</i>. The gray areas indicate the coding regions of the exons. The numbers indicate the nucleotide positions relative to the start codon “ATG”, with “−1” representing the first nucleotide upstream of the start codon encoding “MEIK” or “MQPG” of mouse NBCn2. The arrows indicate the transcription initiation site. The connections between the exons indicate the splicing of <i>Slc4a10</i> transcripts produced from the alternative promoters. Exon 2 is omitted in the mature <i>Slc4a10</i> transcripts produced from P1. (<b>B</b>) Characterization of the distal promoter (P1) expressing mMEIK-NBCn2. (<b>C</b>) Characterization of the proximal promoter (P2) expressing mMQPG-NBCn2. For transcription activity assay, the genomic sequence was subcloned into pGL3 vector containing firefly luciferase reporter gene. The constructs with “R” indicate that the corresponding sequences were subcloned into pGL3 in reverse direction. A plasmid containing the renilla luciferase gene was simultaneously transfected with pGL3 containing the firefly luciferase gene. The ratios of the fluorescence intensity of firefly to that of renilla were used as indices of the transcription activities. Each bar represents the mean of at least three independent experiments. Quadruplicates were prepared for each construct in each experiment.</p

Expression of NBCn2 proteins in rat tissues.

<p>(<b>A</b>) Expression of NBCn2 with the originally described Nt in rat tissues. (<b>B&C</b>) Expression of NBCn2 with the novel Nt in rat tissues. For each tissue, equal amount (50 µg) of membrane proteins were separated on 8% SDS-PAGE, and blotted onto PVDF membrane. Two parallel blots were simultaneously prepared, and probed with anti-MEIK or anti-MCDL at a dilution of 1∶5000.</p

Quantitative PCR analysis of the relative abundances of transcripts encoding mMEIK-NBCn2 and mMQPG-NBCn2 in mouse brain.

<p>(<b>A</b>) Representative amplification plot of qPCR for the transcripts encoding the total of mMEIK-NBCn2 in mouse brain. (<b>B</b>) Representative amplification plot of qPCR for the transcripts encoding the total of mMQPG-NBCn2 in mouse brain. (<b>C</b>) Summary of threshold cycles (C_T) for mMEIK-NBCn2 and mMQPG-NBCn2. The amplification efficiencies were 91.7±2.4% (n = 5) for mMEIK-NBCn2, and 93.1±3.5% (n = 5) for mMQPG-NBCn2, p = 0.76 by student's t-test. The data in panel C represent the summarized C_T for the undiluted cDNA samples obtained from four experiments like those shown in panels A and B. ΔRn: normalized reporter signal of qPCR products subtracted by the background signal. Two-tailed student's t-test was performed for statistical comparison between the amplification efficiencies and C_T values of mMEIK- vs. mMQPG-NBCn2.</p

Diagram of primary structures of NBCn2 variants.

<p>The unique portion (16 aa) of the MEIK-NBCn2 Nt are represented in blue. The unique Nt of novel rat and mouse NBCn2 variants are represented in green. Compared to mouse, the rat novel Nt contains an additional extension of 13 aa residues (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055974#pone-0055974-g002" target="_blank">Figure 2B</a>). Insert A (30 aa, orange) is encoded by exon 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055974#pone-0055974-g001" target="_blank">Figure 1A</a>. The unique long Cts (21 aa, indicated by yellow) of NBCn2-C, -D, -G, and -H contain a PDZ-binding motif “ETCL”. The unique sequences of the two non-PDZ-Ct are indicated at the right end. Ala²⁵⁶ (position 256 relative to the first Met of MEIK-NBCn2) may be omitted in MEIK-NBCn2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055974#pone.0055974-Liu3" target="_blank">[25]</a> as well as the novel NBCn2 variants identified in the present study (i.e., rat NBCn2-F and rat NBCn2-H). The diagram was drawn to scale (scale bar: 100 aa). The sequence alignment was based on human NBCn2-A (accession# NP_071341), human NBCn2-B (#AAQ83632), rat NBCn2-C (#AAO59639), mouse NBCn2-D (#ADX99207), rat NBCn2-E (#AFP48940), rat NBCn2-F (#AFP48941), rat NBCn2-G (#AFP48942), rat NBCn2-H (#AFP48943), mouse NBCn2-I (#AFQ60533), mouse NBCn2-J(#AFN27376).</p

Cloning of novel NBCn2 variants.

<p>(<b>A</b>) Structure of the updated mouse <i>Slc4a10</i> gene. The updated mouse <i>Slc4a10</i> contains 28 exons (represented by vertical bars), among which the 2nd was newly identified in the present study. The white areas of the bars represent the UTRs. The exon numbers are indicated on the top of the bars. The two triangles indicate the two major cassette exons—exon 9 (insert A) and exon 27 (insert B)—that can be alternatively spliced in or out. Arrows a and b indicate the approximate positions of the anti-sense primers used for the 5′-RACE. The diagram was drawn to scale (scale bar: 10 kb). (<b>B</b>) Amplification of 5′-UTR of <i>Slc4a10</i> transcripts by nested 5′-RACE from mouse brain. The 1st lane represents the product of unnested RACE reaction, whereas the 2nd lane represents the nested PCR products. (<b>C</b>) Cloning of the full-length cDNA encoding NBCn2 variants containing the novel Nt from mouse brain tissues. (<b>D</b>) Cloning of the full-length cDNA encoding NBCn2 variants containing the novel Nt from the whole rat brain tissues. Nested RT-PCR was performed to amplify the full-length cDNA. H₂O was used as the template for control.</p