Hybrid r rna purification kit

Mice were anesthetized with isoflurane and 250-µm slices were prepared in PBS, using a vibratome (Leica VT1200). The LH was microdissected bilaterally, and samples were immediately frozen on dry ice and stored at −80 °C before RNA isolation. Total RNA was extracted from dissected samples using a Hybrid-R RNA purification kit (GeneAll Biotechnology). Purified RNA samples were reverse transcribed by using the SuperScript-IV First-strand cDNA synthesis kit (Invitrogen). qPCR was performed by using TaqMan Gene Expression Assay Kit (Applied Biosystems). All TaqMan probes were purchased from Applied Biosystems and are as follows: Lepr (Mm00440181_m1), Hcrtr1 (Mm01185776_m1), Mchr1 (Mm00653044_m1), Hcrt (Mm01964030_s1), Pmch (Mm01242886_g1), Galr1 (Mm00433515_m1), Nts (Mm00481140_m1), Gal (Mm00439056_m1), Cartpt (Mm04210469_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Mm99999915_g1). Target amplification was performed by using ViiA 7 Real-Time PCR System (Applied Biosystems) with QuantStudio Real-Time PCR software v1.3. The relative mRNA expression levels were calculated via a comparative threshold cycle (C_t) method using GAPDH as an internal control: ΔC_t = C_t (gene of interest) − C_t (GAPDH). The gene expression fold change, normalized to the GAPDH and relative to the control sample, was calculated as the 2^−ΔΔCt methods^{64 (link)}.

Total RNA was harvested from RAW 264.7 cells using a Hybrid-R RNA purification kit (GeneAll Biotechnology, Seoul, Korea), according to the manufacturer's guidelines. After the extraction process, the concentration and purity of the isolated RNA were measured at 260 and 280 nm by spectrometry. cDNA was synthesized from total RNA (1 μg) by reverse transcription using the ImProm-II Reverse Transcription System (Promega Corp., Madison, WI, USA), according to the manufacturer's guidelines. The levels of target mRNAs were measured by quantitative RT-PCR in reaction mixtures containing 2 μl cDNA and primer pairs using a KAPA SYBR Fast qPCR kit (Kapa Biosystems Inc., Wilmington, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as a housekeeping gene. The primer pairs used in this study are as follows: gp91phox (NOX2), 5'-CCA ACT GGG ATA ACG AGT TCA-3' (forward) and 5'-GAG AGT TTC AGC CAA GGC TTC-3' (reverse); and manganese-dependent superoxide dismutase (MnSOD), 5'-AAC TCA GGT CGC TCT TCA GC-3' (forward) and 5'-GCT TGA TAG CCT CCA GCA AC-3' (reverse). Relative gene expression was quantified using the ΔΔCt method, and the levels of expression of target genes were normalized against GAPDH expression and reported as fold changes relative to the control group.

Mice were anesthetized with isofluorane and 250 μm slices were prepared in PBS, using a vibratome (Leica VT1200). The LH was microdissected bilaterally, and samples were immediately frozen on dry ice and stored at −80 °C prior to RNA isolation. Total RNA was extracted from dissected samples using a Hybrid-R RNA purification kit (GeneAll Biotechnology). Purified RNA samples were reverse transcribed by using the SuperScript-IV First-strand cDNA synthesis kit (Invitrogen). qPCR was performed by using TaqMan Gene Expression Assay Kit (Applied Biosystems). All TaqMan probes were purchased from Applied Biosystems and are as follows: Penk (Mm01212875_m1), Lepr (Mm00440181_m1), Hcrt (Mm01964030_s1), Pmch (Mm01242886_g1), GR (Nr3c1; Mm00433832_m1) and glyceraldhyde-3-phosphate dehydrogenase (GAPDH; Mm99999915_g1). Target amplification was performed by using QuantStudio 3 Real-Time PCR System (Applied Biosystems) with QuantStudio Real-Time PCR software. The relative mRNA expression levels were calculated via a comparative threshold cycle (Ct) method using GAPDH as an internal control: ΔCt = Ct (gene of interest) – Ct (GAPDH). The gene expression fold change, normalized to the GAPDH and relative to the control sample, was calculated as 2^-ΔΔCt.

Total RNA was extracted from leaf, stem, and root tissues collected at heading time, and from seeds 5 and 20 DAP, using Hybrid-R™ RNA Purification Kit (GeneAll, Korea). All RNA samples were treated with RNase-free Recombinant DNase Ι (Takara Bio, Japan) to eliminate genomic DNA contamination. First-strand cDNA was synthesized using M-MLV reverse transcriptase (Promega, USA). qRT-PCR was performed using SYBR Premix Ex Taq (Takara, Japan) on a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA), according to the manufacturer’s instructions. Primers used for qRT-PCR analysis are listed in Additional file 1: Table S1. Expression levels of LE were normalized relative to UBIQUITIN5, a housekeeping gene.

Cells were grown in LB medium containing 2% (w/v) glucose until mid-exponential growth phase, and total RNA was extracted using Hybrid-R RNA purification kit (GeneAll biotechnology, Korea) according to the manufacturer’s instructions. Quantitative reverse transcription PCR (qRT-PCR) was performed using a Rotor-Gene Q (Qiagen, Germany) and SensiFAST™ SYBR No-ROX One-Step Kit (Bioline, USA). Solutions of 5.0 μL of 2× SensiFAST™ SYBR No-ROX One-Step mix, 0.1 μL of reverse transcriptase, 0.2 μL of RNase inhibitor, 0.4 mM forward and reverse primers (gfpm-qPCR-F and gfpm-qPCR-R), 2.0 μL of isolated total RNA (10 ng/μL), and 1.9 μL of diethylpyrocarbonate (DEPC)-treated water were mixed for each qRT-PCR reaction and qRT-PCR was performed as follow: 45 °C for 10 min, 95 °C for 2 min, and then 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 5 s. The value of ΔΔC_t was averaged from triplicate measurements. The cysG gene encoding siroheme synthase was used as a reference gene and the genes expressed by the wild-type nar promoter were used as calibrators.

Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to investigate immune-related gene expression. A Hybrid-R RNA purification kit and Riboclear plus kit (GeneAll Biotechnology, South Korea) were used for RNA isolation and residual DNA removal from red sea bream intestines. Next, cDNA was synthesized from the isolated RNA using the PrimeScript 1st strand cDNA synthesis kit (Takara, Japan). Gene expression was examined using TB Green Premix Ex Taq (Takara, Japan) on a TP700/760 Thermal Cycler Dice Real Time System (Takara, Japan), and relative expression was calculated using the Thermal Cycler Dice software V5.0× with the 2^-ΔΔCT method and β-actin as a reference gene. The gene-specific primers used for gene amplification are summarized in Table 2.