Purelink rna extraction kit

RNA was extracted from the colonic mucosal scrapings using the Purelink RNA Extraction kit (Invitrogen) and was then submitted for RNA-Seq by Novogene. Fragments per kilobase of transcript sequence per millions base pairs (FPKM) sequenced were analyzed for differential gene expression, principal component, and Gene Ontology (GO) pathway enrichment analysis.

Total RNA was extracted from the pooled samples of upper internodes (internodes 2, 3, and 4, numbered from top to bottom) for Rio, BTx406, and R9188, respectively, using TRIZOL and PureLink RNA extraction kit (Invitrogen). The samples were collected from the plants grown in a split-plot design and are the same samples used for RNA-seq of Rio/R9188/BTx406 as described previously [12 (link)]. The concentration and purity of the RNA were evaluated using a Nanodrop 2000 spectrophotometer. After cDNA synthesis with SuperScript III First Strand kit, real-time quantitative PCR (qPCR) was conducted with PowerUp SYBR Green mastermix (Thermo Fischer) using the ABI StepOne Plus Real-Time PCR system. Relative expression levels were calculated using the ΔΔCT method with Ubiquitin as the internal reference gene because of its stable expression determined by the RNA-seq data [12 (link)]. All real-time qPCR primers are listed in Additional file 8.

RNA was isolated directly from the cells in culture using the PureLink RNA extraction kit (Invitrogen) according to the manufacturer's instructions. A total of 50‐ng RNA was then analyzed in the TaqMAn OneStep RNA to Ct kit (Life Technologies). We used TaqMan primer/probe mix (ABI) for the following genes for mouse/human: Runx2/RUNX2, SP7, Bglap/BGLAP, Col1a1/COL1A1, Dmp1/DMP1, Pparɣ/PPARɣ, Adipoq/ADIPOQ, and Lpl/LPL, which were run on a Quant Studio 3 Real‐Time PCR instrument (Life Technologies). MPC2 genes were normalized to either hypoxanthine guanine phosphoribosyl transferase (Hprt) or VIC‐labeled primer limited Actb; hMSCs were normalized to VIC‐labeled primer limited ACTB and calculated using the 2^‐Δ/ΔCt calculation method using day 1 controls as baseline. Primer/probe information is available upon request.

Scrapings of the mouse colonic mucosa were homogenized with a rotor-stator homogenizer in RNA lysis buffer containing 1% β-mercaptoethanol. RNA was isolated using the Purelink RNA Extraction kit (Invitrogen) following manufacturer’s instructions. cDNA was prepared by the iScript Reverse Transcription Supermix for qPCR using 1 μg RNA and diluted by 80 μL nuclease-free water. qPCR was performed using PowerUp SYBR Green Master Mix (Invitrogen) with 20 ng cDNA added to each reaction. Predesigned forward and reverse primer sets were purchased from Integrated DNA Technologies (IDT) and used at a final 500 nM concentration. qPCR was performed using the QuantStudio3 Real-Time PCR System (Applied Biosystems) with the following cycling conditions: 2 minutes at 50°C, 2 minutes at 95°C, denaturing step for 1 second at 95°C, extension and annealing for 1 minute at 60°C, and a dissociation melt curve stage to confirm primer specificity. The results are expressed as fold-increase mRNA expression of the gene of interest normalized to 18S expression by the ΔΔCt method.

of precleared RNA per sample were fragmented with RNA fragmentation buffer (100 mM Tris, 2 mM MgCl₂) for 3 min at 95 °C and placed on ice immediately after heating. 10% of RNA was kept as input. One microgram of m⁶A antibody (Abcam, #ab151230) and control antibody (IgG, Santa Cruz Biotechnologies, Dallas, USA, #sc‐2025) were coupled to agarose A beads (GE Healthcare, Chicago, USA) in a rotation wheel for 1 h at 4 °C. After incubation, beads were washed twice in reaction buffer (150 mM NaCl, 10 mM Tris‐HCl, 0.1% NP‐40). RNA was added to the antibody‐coupled beads and incubated for 3 h at 4 °C in a rotating wheel. Subsequently, beads were washed 3X in reaction buffer, 3X in low salt buffer (50 mM NaCl, 10 mM TrisHCl, and 0.1% NP‐40), and 3X in high salt buffer (500 mM NaCl, 10 mM TrisHCl, and 0.1% NP‐40). After the last wash, beads were resuspended in Lysis buffer and RNA was extracted using the PureLink RNA extraction kit (Invitrogen, Carlsbad, USA, #12 183 016).

Total RNA was extracted from liver samples using the Purelink RNA extraction kit (Invitrogen) according to the manufacturer’s protocol. The concentration and purity of the total RNA samples were obtained by using a Nanodrop ND-1000 spectrophotometer. cDNA was synthesized using the Maxima first-strand kit (Invitrogen) according to the manufacturer’s recommendations. The synthesized cDNA samples were stored at −20 °C until further use.

Purelink RNA Extraction Kit (Invitrogen) was used to isolate RNA from samples. RNA (1 μg) was then converted to cDNA using SuperScript Reverse Transcriptase III (Invitrogen) according to the supplier’s instructions. Real-time quantitative PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) according to QuantStudioTM 12 K Flex Real-Time PCR System protocol. GAPDH mRNA level was used to normalize samples.