Fastpure rna kit

Total RNAs were extracted using either an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) or a FastPure® RNA Kit (Takara Bio, Shiga, Japan). First-strand cDNA synthesis was performed using PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara Bio) with oligo-dT primers provided by the manufacturer. Procedures for cDNA cloning are described in the Supplementary information. Briefly, partial fragments of targeted genes for SrPK, SrPEPtase, SrPEPC, and SrPEPCK were first amplified using PCR with Takara Ex Taq® (Takara Bio) and the primers are listed in Supplementary Table 2. Gene-specific primers were then designed to perform 5′- and 3′-rapid amplification of cDNA ends using SMARTer™RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). To isolate full-length cDNAs, final PCR amplifications were performed with the KOD -Plus- (Toyobo, Osaka, Japan) with the primers shown in Supplementary Table 2. The obtained fragments were cloned into the T-Vector (pMD19, Takara Bio) and sequenced in both directions. Full-length sequences were determined by isolating at least 2 independent clones with identical sequences. DNA sequences were analyzed using GENETYX software (Genetyx, Tokyo, Japan). Complete cDNAs encoding SrPEPC, SrPK, SrPEPCK, and SrPEPtase were deposited in the DNA Data Bank of Japan with Accession numbers LC155943, LC155944, LC155945, and LC155946, respectively.

Total RNA was extracted from DRG and spinal cord samples using a Fastpure RNA kit (Takara Bio, Otsu, Japan) and treated with DNAse (Turbo DNAse, Ambion, Austin, TX, USA) prior to reverse transcription. One microgram of total RNA was reverse-transcribed into cDNA using SuperScript III (Thermo Fisher Scientific Inc., Waltham, MA, USA) and an oligo dT adapter primer. Specific BNP cDNA was amplified by PCR using Ex Taq HS DNA polymerase (Takara Bio) and the following rat BNP primers: 5′-CAGAAGGTGCTGCCCCAGATG-3′ (sense) and 5′-GACTGCGCCGATCCGGTC-3′ (antisense). The amplification profile consisted of an initial step of denaturation at 95°C for 1 min, 36 cycles of 10 s at 98°C, 30 s at 55°C and 1 min at 72°C, followed by final extension at 72°C for 8 min. The PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide.

The urine samples were centrifuged and the pellet cells were washed twice with PBS. Next, RNA extraction was carried out using the FastPure RNA kit (Takara Bio, Inc., Otsu, Japan). About 1 µg of total RNA was subjected to reverse transcription using the PrimeScript RT reagent kit (Takara Bio) according to the manufacturer’s instructions.

Total RNA was isolated from the intestinal tissue samples collected in trials B and C according to FastPure RNA kit (TaKaRa Bio Inc., Shiga, Japan). All other procedures were in agreement with the manufacturer’s protocol. RNA purity and integrity were evaluated by Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) just before real-time quantitative PCR (RT-qPCR) analysis.
The expression of OR51E1 gene was quantified by RT-qPCR. One microgram of total RNA was reverse-transcribed using the ImProm-II Reverse Transcription System (Promega, Milan, Italy). Primers were designed based on a specific porcine nucleic acid sequence (Gen-Bank database) using Primer 3 version 0.4.0 (http://frodo.wi.mit.edu/primer3/). The RT-qPCR reactions were performed in a LightCycler Real-Time PCR Systems (Roche Applied Science, Bazel, Switzerland) by a shuttle PCR (2 steps) following the procedure described by Trevisi et al. [28 (link)]. The expression of data was normalized by geometric mean of expression data for two housekeeping genes: hydroxymethylbilane synthase (HMBS) and ribosomal protein L4 (RPL4) genes for gastric tissue samples (study B), and HMBS and TATA box binding protein (TBP) genes for jejunal tissue samples (study C), following Nygard et al. [29 (link)]. Primers and amplification conditions for OR51E1 and housekeeping genes are reported in Table 1.

Total RNA was isolated from the gastric tissue samples using the FastPure RNA kit (TaKaRa Bio Inc., Shiga, Japan). All other procedures were in agreement with the manufacturer’s protocol. The RNA purity and integrity were evaluated using Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) just before the transcriptome analysis. The total RNA was hybridized on Affymetrix Porcine Gene 1.1 ST array strips. The hybridized arrays were scanned on a GeneAtlas imaging station (Affymetrix, Santa Clara, CA, USA). Performance quality tests of the arrays including the labelling, hybridization, scanning and background signals using Robust Multichip Analysis were carried out on the CEL files using the Affymetrix Expression Console.

To confirm the effects of the SNP in the CYP19A1, the expression levels of CYP19A1 gene in noncancerous lung were quantified by real-time RT-PCR. The FastPure RNA Kit (Takara Bio, Shiga, Japan) was used to isolate total RNA from the frozen tissue. Levels of CYP19A1 mRNA were measured by real-time RT-PCR. Total RNA (1μg) was reversed transcribed using the SuperScript III First-Strand System (Life Technologies Inc., Rockville, MD, USA). Quantitative RT-PCR reactions were performed on the ABI ViiA^™ 7 instrument using TaqMan^® Universal Master Mix and gene-specific primer mixes (both from ABI): CY19A1 (Hs00903413_m1), The Ct values for each gene were normalized to the housekeeping gene GAPDH (Hs02758991_g1), and the fold change in the transcript level was caluculated using the ΔΔCt method. Expression levels were calculated relative to those from the lung AD cell line H358.