Quantitative PCR determination of spR-12 in somatic and germ line cells.

*<p>Expression relative to that of miR-16.</p>**<p>Expression relative to actual number of cells. Serial dilutions of synthetic spR-12 RNA were used to generate a standard curve of the log of template quantity versus threshold cycle (C_(t)). The concentration of RNA ranged from 10–10⁶ initial copies. A minimum of eight oocytes and/or embryos was used in each experiment. Each value corresponds to three or four independent experiments, performed in duplicate. The analysis for spR-13 was not done as it was not possible to synthesize the spR-13 RT-primer.</p

Oligonucleotides used in this study.

<p>For the oligonucleotide, RTQ primer, V represents the nucleotides A, C, or G; N indicates that the nucleotide is equivalent to A, C, G, or T.</p

Illumina GA sequencing reads of spR-12 sequences and variants.

<p>Bold type letters show nucleotide sequences which are not mapped on the genome.</p

Search for double-stranded piRNA precursors of spR-12 and -13 by strand-specific RT-PCR.

<p>Total RNA from adult mouse testes and epididymis was reverse-transcribed with a primer with an antisense sequence, with a primer with a sense sequence, and with no primer. PCR was conducted using gene-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone-0044542-t005" target="_blank">Table 5</a>). The product was 301 bp for the spR-12 locus, and 351 bp for the spR-13 locus, whose nucleotide sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone-0044542-g007" target="_blank">Figure 7</a>. LINE 1 and β-actin were used as positive and negative controls, respectively.</p

Expression profiling of spR-12 and -13 and the proximal piRNAs.

<p><b>A.</b> RT-PCR analysis of spR-12 and -13 and their flanking piRNAs in different mouse tissues. The piRNAs adjacent to spR-12 (mmu_piR_032165) and -13 (mmu_piR_037961) and the spR-12 and -13 sequences are indicated in red and blue, respectively. Numbers are the nucleotide coordinates of the genomic DNA. Primers specific to each small RNA are shown as blacks arrows. RT-PCR was performed with a specific primer and RTQ-UNIr universal primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone-0044542-t005" target="_blank">Table 5</a>). The PCR products were analyzed on 3% (w/v) agarose gels and contained the expected 120 bp fragment. The positive control was let-7a. <b>B.</b> Short RNA reads mapped in the spR-12 locus and the adjacent piRNA mum_piR_032165. X axis indicates the genomic coordinates and Y axis the number of sequence reads. Location of the spR-12 and piRNA sequences is indicated with the genomic coordinates and number of read counts.</p

Size distribution of testis and sperm RNAs.

<p>Electropherograms were obtained using the RNA 6000 Nano Chip. <b>A.</b> RNA prepared from mouse sperm contained no or very low levels of long RNAs, including ribosomal RNAs (18S- and 28S-rRNA) compared with the total RNA from mouse testis <b>(B)</b>. <b>C.</b> Classification of all sequence reads in the mouse sperm total RNA library.</p

Sperm/testis ratio of read counts of spR-12 and flanking-spR-12.

<p>Read counts of each sequence were normalized to reads per million (RPM).</p

Novel small RNAs from the mouse sperm identified by deep sequencing.

a<p>(+): plus strand, (−): minus strand.</p>b<p>piRNA locus: mapped in piRNA locus.</p>c<p>Stem-loop: potential pre-miRNA like secondary structure.</p>d<p>RT-PCR: validated by poly(A)-tailed RT-PCR.</p

Expression analysis of thirteen sperm small RNAs. A.

<p>Poly(A)-tailed RT-PCR validation of 13 RNAs identified by deep sequencing. RNAs isolated from adult testis and sperm were polyadenylated. Reverse transcription was carried out using an RTQ primer, with or without reverse transcriptase. The cDNAs were amplified by PCR using a primer specific to each small RNA and an RTQ-UNIr universal primer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone-0044542-t005" target="_blank">Table 5</a>). The expected cDNA sizes for the RNAs were approximately 120 bp. The PCR products were electrophoresed on 3% (w/v) agarose gels and stained with ethidium bromide. The positive control was let-7a. <b>B.</b> spR-12 and -13 nucleotide sequences. <b>C.</b> Detection of spRs by Northern blot hybridization. Lanes 1 and 3: synthetic oligoribonucleotides with the sequences of spR-12 and -13, respectively; lanes 2 and 4∶1 µg of total sperm RNA; lanes 1 and 2 hybridized with spR-12-antisense probe, lanes 3 and 4 with spR-13-antisense probe (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone-0044542-t005" target="_blank">Table 5</a>). <b>D.</b> Analysis of posttranscriptional modification of spRs termini. Northern blot analysis of testis, sperm samples and synthetic spRs, untreated (−) or treated (+) with the oxidation and β-elimination reagents. Only RNAs having both 2′ and 3′ hydroxyl groups react with NaIO₄; β-elimination shortens a NaIO₄-reacted RNA by one nucleotide, leaving a 3′ monophosphate terminus. NaIO₄-reacted (β-eliminated) RNAs migrate faster in polyacrylamide gel electrophoresis than does the original untreated RNA. Both spRs, flanking-spR-12 (mmu_piR_032165) and piR-1 (mmu_piR_030365) lack either a 2′ or 3′ hydroxyl group, because they failed to react with NaIO₄.</p

Predicted secondary structures of spR-12 and -13 regions.

<p>Positions in the mouse genome used for this analysis were chr17∶27455400–27455700 for spR-12 and chr17∶27496250–27496600 for spR-13. Position of the cDNA read sequences are shown by the blue arrows. The structure shown is the most stable configuration predicted by ‘RNAfold’ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044542#pone.0044542-Zuker1" target="_blank">[20]</a>.</p