Purelink rna purification kit

The constructs were linearized by AgeI (MHC-ftz-Δi-V5-His and MHC-ftz-Δi-V5-His-GU→AC) or XhoI (MHC-ftz-Δi) digestion and precipitated overnight in -20°C with 40 mM KOAc and 2.5X 100% ethanol. In vitro transcription and capping reactions were performed using the T7 mMESSAGE mMACHINE transcription kit (Ambion) following the manufacturer’s protocol. The resulting RNA was polyadenylated using the Poly(A) tailing kit (Ambion) to generate poly(A) tails of 200 to 300 nucleotides in length. The in vitro synthesized RNA was then purified with Purelink RNA purification kit (Ambion), eluted with 20 μl RNase free water and resuspended accordingly in 10X injection buffer.

TvGv29-8 and TaT6776 conidia were inoculated in PDB at a final concentration of 10⁷ conidia/ml and incubated at 23–25°C under illumination of 16 h light/8 h dark cycles, using daylight tubes 24 W/m², 9,000 lx. After 27 h, fungal RNA was isolated and purified using the PureLink RNA purification kit (Ambion, Thermo Scientific). To avoid genomic DNA (gDNA) contaminations, the RNAs were treated with DNase I (Ambion, Thermo Scientific), and complementary DNA (cDNA) synthesis from messenger RNA (mRNA) (200 ng) was performed using cDNA first-strand synthesis kit (Thermo Fisher) with hexamer primer according to manufacturer’s instruction.
Reactions were performed using the Real-Time PCR PowerUp SYBR Green Master Mix kit (Applied Biosystems) in the ViiA7 Real-Time PCR system (Applied Biosystems). Each RT-PCR reaction was performed in three biological and technical replicates and a no-template control. To determine the specificity of the primer pairs used in the current study, 1.5% agarose gel electrophoresis and melt curve analyses were performed (Supplementary Table S2). To determine the PCR efficiency of each primer pair, a standard curve was generated using linear regression, and the Cq slope was calculated based on the Cq values for all dilutions (5 points, 10-fold dilutions from 50 to 50,000).

RNA was purified from adult zebrafish tissues or 10-30 pooled embryos at 3 or 4 dpf in TriZol (Sigma-Aldrich; T9424) using the PureLink RNA purification Kit (Ambion). DNase treated RNA was reverse transcribed with a polyT₍₂₃₎ primer using Protoscript II RT-PCR kit (NEB; M0368S). Target genes were amplified in triplicate from cDNA by qRT-PCR with 1 µM primers (Table S1). Standard curves were generated to confirm primer efficiencies. Target gene expression was normalized to beta-actin for comparison by the ΔΔCt method. Three or four biological replicates were used for each treatment for statistical comparisons.

Dispersed islets were either transduced as above or left untransduced and cultured in RPMI media supplemented with the cell stressors described above for 36 h. RNA was isolated using Trizol and the PureLink RNA purification kit (Ambion, Burlington, ON, Canada). cDNA was generated using Superscript III (Invitrogen, Burlington, ON, Canada). Taqman probes were used to quantify β-actin and Myt3, all other primers were designed using Primer3plus and ordered from IDT. A Viia7 real-time PCR system and SYBR Green supermix (Applied Biosystems, Burlington, ON, Canada) or Taqman Fast Advanced Master Mix (Applied Biosystems) was used for all reactions. cDNA (10ng) was used in each reaction, with all reactions done in triplicate. β-Actin was used as an internal control and the change in expression was calculated using 2^−ΔΔCt.

Total RNA was prepared from cells using the Purelink RNA purification Kit (Invitrogen, Carlsbad, CA, USA). cDNA was prepared from 1 μg of total RNA using the QuantiTect RT Kit (Qiagen, Venlo, the Netherlands). Quantitative RT-PCR was performed using TB Green Premix ExTaq II (Takarabio, Otsu, Japan) and a LightCycler96 (Roche Basel, Switzerland). Primers used for qPCR are listed in Supplementary Table S1.

Following two washes of both the apical and basolateral surfaces of polarized Calu-3 cells with sterile Dulbecco’s phosphate buffered saline (D-PBS), total cellular RNA was extracted from Calu-3 cells using PureLink^® RNA purification kit (Invitrogen) per the manufacturer’s protocol, and stored at −20 °C until use. Expression of the RSV matrix (M) gene was determined using primers (forward, position 3257-3282) 5′-GGC AAA TAT GGA AAC ATA GCT GAA-3′ and (reverse, position 3312-3340) 5′-TCT TTT TCT AGG ACA TTG TAY TGA ACA G-3′) [51 (link)]. Briefly, real-time quantitative RT-PCR was performed on a Stratagene Mx3000 detection system (Agilent Technologies, Santa Clara, CA, USA). Threshold cycles (C_t) for each sample were calculated and serial dilutions of known PFUs of RSV A2 RNA were used to obtain a standard curve, from which the relative amount of genome, in PFU/mL, was determined for each sample. Commercially available TaqMan probe mixes were used to determine IFNα1, IFNλ, suppressor of cytokine signaling (SOCS-1) and SOCS3 expression profiles relative to 18S rRNA (Applied Biosystems, Foster City, CA, USA).

At all HPOs and HPSs, 1 g of the unfertilized eggs was placed in cryotubes, frozen in liquid nitrogen and then stored at -80°C freezer until RNA isolation. Samples were collected in three replicates for each fertilization time.
To prepare total RNA, an equal weight of 20 eggs was taken from each tube and treated with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) in Precellys24 homogenizer (Bertin Instruments), 5500 rpm for 2×20 sec with a 5 sec interval. RNA was isolated using the PureLink RNA Purification Kit (Invitrogen) according to the manufacturer’s instructions. During RNA isolation, samples were depleted of genomic DNA using a PureLink DNase Kit (Invitrogen) according to the manufacturer’s protocol. The RNA concentration and RNA purity were assessed using a NanoDrop Spectrophotometer (ND-1000, Thermo Scientific).
A concentration of 1000 ng RNA was used to generate cDNA using TaqMan reverse transcription Reagents (Life Technologies). Random hexamers were used to prime the reaction. Reverse transcription was performed at 25°C for 10 min, at 48°C for 1 hour and finally 95°C for 5 min. Control reactions were run without TaqMan reverse transcriptase and used as negative controls in the real-time PCR study.