Purelink micro to midi total rna purification system

A. baumannii cultures were grown to log phase (OD₆₀₀ 1.00) in Muller Hinton broth with shaking at 37°C before RNA extraction. Total RNA was isolated from cells using the PureLink™ Micro-to-Midi Total RNA Purification System (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA quantity and quality were assessed using a BIOANALYZER 2100 (Agilent Technologies Inc., Germany), followed by RNA-Seq. The RNA integrity number (RIN) of total RNA should be greater than 8.0, and rRNA ratio (23S/16S) should be greater than 1.2.
The RNA-sequencing library was prepared as previously described
[28 (link)]. The constructed sequencing libraries were sequenced using the Illumina HiSeq 2000 platform at Beijing Genome Institute (BGI, Shenzhen, China).

The Rubus sp. var. Lochness plants used in this study were planted at production fields of the company Agricola El Bosque (Lucena del Puerto, Huelva, Spain). Plants and greenhouses were kindly provided by the company and all were handled according to regular agricultural practices [42 (link)]. Plants were grown under “winter cycle” that is, after an artificial cold period, from July to November 2013 under natural light conditions.
Prior to RNA extraction, samples were removed from the −80°C freezer and ground to a fine powder with liquid nitrogen using a sterilized mortar and pestle Total RNA was isolated from red fruits with PureLink™ Micro-to-Midi Total RNA Purification System (Invitrogen™), and after DNase treatment and confirmation of RNA integrity using a triple check, Nanodrop™, Experion™ Automated Electrophoresis System, and gel electrophoresis, the total RNA was used in mRNA preparation, fragmentation and cDNA synthesis.
Beads with oligo(dT) were used to isolate poly(A) mRNA from total RNA (Qiagen GmbH, Hilden, Germany).

Total RNA was extracted using TRIzol® Reagent followed by the PureLink™ Micro-to-Midi Total RNA Purification System according to the manufacturer’s protocol (Invitrogen, USA). Total RNA was quantified based on the A260/A280 absorbance using the Nanodrop ND1000 (Thermo scientific, USA). Total RNA was reversed transcribed using iScript Reverse Transcription Supermix for RT-PCR (Bio-Rad, USA).

Total RNA was prepared using the PureLink Micro-to-Midi total RNA Purification System (Invitrogen, Carlsbad, CA; #12183018A), according to the manufacturer’s protocol. Samples were first treated with DNase I Amp Grade (Invitrogen, Carlsbad, CA; #18068015) to eliminate genomic DNA contamination. RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA; #4368813) according to the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) reactions were performed as described using the Universal Probe Library system (Roche, Basel, Switzerland). Actin and tubulin predeveloped TaqMan assays (Applied Biosystems, Foster City, CA) were used to control for cDNA quantity. qRT-PCR assays were performed on the LightCycler 480 System (Roche, Basel, Switzerland). The primers and probes were as follows: Human actin, F 5′-CCAACCGCGAGAAGATGA, R 5′-TCCATCACGATGCCAGTG, UPL probe #64; Human tubulin, F 5′-CTTCGTCTCCGCCATCAG, R 5′-TTGCCAATCTGGACACCA, UPL Probe #58; Human IL-6, F 5′-GCCCAGCTATGAACTCCTTCT, R 5′-GAAGGCAGCAGGCAACAC, UPL Probe #45; and Human p16^INK4a, F 5′-GAGCAGCATGGAGCCTTC, R 5′-CGTAACTATTCGGTGCGTTG, UPL Probe #34.

Lysis and homogenization of fresh soft tissue were performed before RNA extraction; PureLink Micro-to-Midi Total RNA Purification System (Invitrogen, Cat. No 12183-018, Carlsbad, California, United States) was used. RNA extraction was done using the Purelink RNA Mini Kit (Ambion, Cat. No. 12183-018A, Carlsbad, California, United States). DNase was not used before proceeding with complementary DNA (cDNA) synthesis as recommended in the protocol. For cDNA synthesis, MINT (Evrogen, Cat. No SK001, Moscow, Russia) was used; this kit enzymes synthesize full-length-enriched double-stranded cDNA from a total polyA⁺ RNA. The cDNA was then amplified by 16–21 cycles of polymerase chain reaction and purified using a PCR Purification Kit (Invitrogen K3100-01 Carlsbad, California, United States).

Standard techniques were used. Briefly, the total RNA extraction was undertaken using the PureLink™ Micro-to-Midi Total RNA Purification System (Invitrogen, Carlsbad, CA, USA). The PureLink™DNase kit (Invitrogen) was then used to remove genomic DNA. The integrity of RNA was assessed via agarose gel electrophoresis via the Experion automated electrophoresis station and the Experion StdSens Analysis kit according to the manufacturer’s instructions (Bio-Rad Laboratories Inc., Hercules, CA, USA), and total RNA was quantified using a spectrophotometer (Nanodrop ND-1000, Thermo Fisher Scientific, Newark, DE, Waltham, MA, USA) with readings taken at 260 and 280 nm.

RNA was extracted from joints of uninfected and 21 day-infected WT, FcεR_γ^−/−, MyD88^−/−and double knockout mice using TRIzol® and the PureLink™ micro-to-midi total RNA purification system (Invitrogen, Carlsbad, CA). The quality of RNA was verified in all samples with A₂₆₀:A₂₃₀ ratios greater than 1.7, and A₂₆₀:A₂₈₀ ratios between 1.8 and 2.0. RNA was combined from 4 mice in each group and was converted to complimentary DNA (cDNA) and real-time PCR was performed on the samples using the RT² First Strand Kit and the RT² qPCR Master Mix according the manufacturer's instructions (SABiosciences, Frederick, MD). The master mix containing the cDNA was loaded onto the RT² Profiler PCR Array for Inflammatory Cytokines and Receptors (SABiosciences, PAMM-011), and the amplification was performed according to the manufacturer's protocol using a Bio-Rad iCycler. The data were analyzed using the ΔΔCt method by the Keck facility (Yale University).