Ultrahyb ultrasensitive hybridization buffer

Preparation of the RNA probes and in situ hybridization were carried out as previously described [8 (link)]. Digoxigenin (DIG)- and fluorescein-labeled antisense and sense RNA probes for lamprey parapinopsin and parietopsin mRNAs were synthesized using the DIG RNA-labeling kit and fluorescein RNA-labeling kit (Roche), respectively. Sections were pre-treated with proteinase K and hybridized with each RNA probe in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). The pineal sections hybridized with DIG-labeled probes were incubated with horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche) and subsequently treated with the TSA plus DNP (HRP) system (Perkin Elmer), followed by incubation with Alexa 488-conjugated anti-DNP antibody. The fluorescein-labeled probes were detected through incubation with alkaline phosphatase-conjugated anti-fluorescein antibody (Roche) followed by a color reaction using the HNPP Fluorescent Detection Set (Roche).

Validation of ncRNA was done by northern blotting. Total RNA (15–20 μg/per lane) was resolved on a 8 % denaturing polyacrylamide gel containing 8 M urea for 75 min at 300 V. The RNA was electroblotted onto nylon membrane (BrightStar plus, Ambion) using the Trans-Blot Turbo transfer system (BioRad). After UV-crosslinking in an ultraviolet crosslinker (CL1000, UVP Inc), the membranes were stained with 0.03 % methylene blue in 0.3 M sodium acetate to visualize the RNA bands and control the transfer. Then the membranes were pre-hybridized in Ultrahyb ultrasensitive hybridization buffer (Ambion) for 1 h at 68 °C. Riboprobes were synthesized using the mirVana miRNA Probe construction kit (Ambion) and labelled with [α-32P]-UTP. Labelled riboprobes complementary to each ncRNA target (Additional file 8) were added and incubated at 68 °C overnight. After washing twice in low stringency buffer (Ambion) for 15 min and once with high stringency buffer (Ambion) for 15 min, the membranes were exposed to phosphorimaging screens and the screens were scanned using a phosphorimager 445 SI (Amersham). Decade marker (10–150 nucleotides, Ambion) and RNA century marker (100–500 nucleotides, Ambion) were used as size standards.

Total RNA was isolated from HeLa cell pellets using the RNeasy Mini kit (Qiagen). The RNA was then subjected to electrophoretic separation using a 1% (wt/vol) agarose-formaldehyde (0.75% v/v) gel after which it was transferred to a positively charged Hybond membrane (GE healthcare). The membrane was UV irradiated in order to fix the RNA. ³²P-labeled DNA probes for Skp2 and GAPDH were prepared using the Random primer DNA labeling system (Invitrogen) using restriction digest fragments of Skp2 and GAPDH cDNA. Hybridization was performed by incubating the membrane in ULTRAhyb® Ultrasensitive Hybridization Buffer (Ambion) containing the probe at 42 °C for 16 hours. After washing bands were visualized by autoradiography and quantification performed by phosphorimaging analysis.

For northern blotting, RNA samples were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For northern blotting of small RNA, 50 μg of total RNA was resolved by 15–19% polyacrylamide gel electrophoresis (PAGE) in 1× TBE at 80 V for 4–6 h and transferred to a Hybond membrane (Amersham Biosciences, GE Healthcare) in 0.5× TBE overnight at 28 mA. The UV cross-linked membrane was hybridized in ULTRAhyb® Ultrasensitive Hybridization buffer (Ambion, Austin, TX, USA) using probes of 3′ biotin-labeled DNA oligos (TaKaRa, Otsu, Japan) antisense to the mature miRNA or U6 transcripts (Supplementary Table S3). RNA gel blot analysis was performed according to the methods of Liu et al. (34 (link)).
For real-time PCR, total RNA was treated with DNase I (TaKaRa), followed by a phenol/chloroform extraction to remove contaminating DNA. Approximately 4 μg of purified RNA was used for first-strand complementary DNA (cDNA) synthesis using PrimeScript® Reverse Transcriptase (TaKaRa) with oligo(dT) primers. Real-time PCR was performed using the specific primer pairs (Supplementary Table S3) in the MyiQ2 Two-color Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). The comparative threshold cycle method was used to determine relative transcript levels. Quantitative PCR for each gene was done with at least three biological replicates.