RNA was extracted using a combination of TRIzol (Life Technologies) and RNAqueous-Micro (Ambion) technologies. Briefly, the tissue was homogenized in 1 ml of TRIzol reagent per 100 mg of tissue. For phase separation, 0.2 ml of chloroform per 1 ml TRIzol was added to the mixture, and samples were incubated at room temperature for 2 min. Samples were then centrifuged at 12,000×g for 15 min. Following centrifugation, the supernatant was removed and column purified using the standard RNAqueous-Micro protocol (Ambion). RNA concentration and purity were then initially determined using a Nanodrop 1000 Spectrophotometer (Thermo).
Rnaqueous micro
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RNAqueous-Micro kit is a tool for the isolation and purification of total RNA from small samples. It is designed to work with sample sizes ranging from a few cells to 1 milligram of tissue.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Cfx96 c1000 thermal cycler, by Bio-Rad (2 mentions)
Mangomix, by Meridian Bioscience (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Rq1 rnase free dnase, by Promega (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
As lmd, by Leica Microsystems (2 mentions)
Fam tamra, by Eurogentec (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Abi prism 7000, by Thermo Fisher Scientific (1 mentions)
First strand synthesis system for rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini, by Qiagen (1 mentions)
Rnasezap, by Thermo Fisher Scientific (1 mentions)
Rneasy micro, by Qiagen (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
16 protocols using rnaqueous micro
1
RNA Extraction Using TRIzol and RNAqueous
2
Quantification of EPHX2 mRNA Levels
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Levels of EPHX2 (sEH gene) mRNA were determined as previously described [8] . In brief, RNA was isolated using a commercial kit (RNAqueous-Micro, Ambion, Austin, TX), contaminant genomic DNA removed by DNase treatment, and RNA reverse transcribed using a commercial high capacity cDNA archive kit (Applied Biosystems, Carlsbad, CA). Resulting cDNA was amplified using TaqMan Universal PCR amplification in a commercial sequence detection system (ABI Prism 7000, Applied Biosystems, Carlsbad, CA). Quantitative PCR was performed in a 96-well plate using 50 µl total volume, in triplicate. PCR was concurrently run on controls without template to assess DNA contamination and primer-dimer formation. Remaining RNA not reverse transcribed was included to control for genomic DNA amplification. 18S was measured as an internal control using an 18S mRNA control kit (FAM-TAMRA, Eurogentec, Seraing, BEL). EPHX2 primers were purchased as a TaqMan Gene Expression Assay (Invitrogen, Catalog #4351372). All final EPHX2 mRNA levels were normalized to 18S.
3
Total RNA Extraction and cDNA Synthesis from Gut Tissue
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Total RNA from the gut tissue was extracted with the Trizol reagent (Invitrogen). RNA was incubated with 1 U/μg of RQ1 RNase-free DNase for 30 min, at 37 °C. The total RNA of guts was extracted using RNAqueous®-Micro (Ambion), which allows for a better RNA yield from small tissue samples. After purification, the RNA concentration was measured with a Nanodrop® spectrophotometer (Thermo Scientific) and RNA quality was checked using agarose gel electrophoresis. Reverse-transcription into the first strand cDNA was carried out using the First Strand Synthesis System for RT-PCR kit (Invitrogen).
4
Genotyping and qRT-PCR Analysis of Lis1 in Mice
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For genotyping by PCR, the reaction mixture contained MangoMix (Bioline), genomic DNA and 0.5 μM of each primer. PCR conditions for genotyping were as follows: 3 min at 94°C, followed by 35 cycles at 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min. RNA was isolated using RNAqueous-Micro (Ambion) or RNeasy Mini kit (Qiagen). cDNA was prepared from equal amounts of RNAs using Superscript II reverse transcriptase (Invitrogen). Quantitative real-time PCRs were performed using iQ SYBR Green Supermix (Bio-Rad) on a CFX 96 C1000™ Thermal cycler (Bio-Rad). Results were normalized to the level of β2 microglobulin or TATA-binding protein. Mouse Lis1 (Mm01253377_mH) gene levels were analyzed with TaqMan Gene Expression Assays. All primer sequences are listed in Supplementary Table 2 .
5
Total RNA Extraction from Insect Tissues
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Total RNA from whole larvae or carcasses was extracted with TRIzol reagent (ThermoFisher Scientific) following the manufacturer’s instructions. RNA was incubated with 1 U/µg of RQ1 RNase-free DNase (Promega) for 30 min at 37 °C. Total RNA from bacteriomes, guts and fat body was extracted using RNAqueous Micro (Ambion), which allows for a better RNA yield from small tissue samples. After purification, the RNA concentration was measured with a Nanodrop spectrophotometer (ThermoFisher Scientific) and RNA quality was checked using agarose gel electrophoresis. Reverse-transcription into the first strand cDNA was carried out using the iScript cDNA Synthesis Kit (Bio-Rad).
6
RNA Extraction Comparison for LCM Samples
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The following RNA extraction kits were used to isolate total RNA from LCM acquired material: RNeasy® Micro (Qiagen, Germany), miRNeasy Mini (Qiagen, Germany), Arcturus® Picopure® RNA isolation kit (Arcturus, Applied Biosystems, USA.), mirVana™ miRNA isolation kit (Ambion, USA.) and RNAqueous®-Micro (Ambion, USA.). Samples were taken from a −80 °C freezer and lysis buffer from the according RNA extraction kit was immediately added to the sample at room temperature. Buffer and tissue were mixed for 30 s through pipetting and left on the bench for 5 min to assure proper lysis. Extraction was performed according to manufacturer’s protocols with an on-column DNAse digestion step (RNase-Free DNase set, Qiagen, Germany) added after the first wash buffer step for each kit. RNA quality and quantity was measured in duplicate with a 2100 Bioanalyzer and the RNA 6000 Pico Kit according to manufacturer’s protocol (Agilent Technologies, USA.). An additional water wash step of the Bionalyzer electrodes was added prior to PicoChip analysis to minimize RNaseZap (Applied Biosystems, USA.) associated ghost peaks.
7
Genotyping and qRT-PCR Analysis of Lis1 in Mice
8
Enrichment of non-trans-spliced mRNAs
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Total RNA from each stage of development was isolated using RNAqueous Micro (Ambion) and treated by TerminatorTM 5’-Phosphate-Dependent Exonuclease to deplete excess small RNAs. A modified CAGEscan protocol [11 (link)] was carried out at DNAFORM, Yokohama City, Japan. The standard CAGEscan protocol was modified in order to separate trans-spliced from non-trans-spliced transcripts by first using a custom designed 5’ linker, specific to the 5’ spliced leader sequence, before using standard linkers for non-trans-spliced mRNAs. Sequenced libraries for each stage therefore included only non-trans-spliced transcripts.
9
Total RNA Extraction and cDNA Synthesis
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Total RNA from whole larvae and carcasses was extracted with TRIzol reagent (Invitrogen) following the manufacturer’s instructions. RNA was incubated with 1 U/μg of RQ1 RNase-free DNase (Promega) for 30 min at 37 °C and purified using Nucleospin RNAClean-up (Macherey-Nagel). Total RNA from bacteriomes was extracted and purified using RNAqueous Micro (Ambion), which allows for a better RNA yield from small tissue samples. After purification, the RNA concentration was measured with a Nanodrop® spectrophotometer (Thermo Scientific), and RNA quality was checked using agarose gel electrophoresis. Reverse transcription into the first strand cDNA was carried out using the iScript™ cDNA Synthesis Kit (Bio-Rad).
10
Isolating and Analyzing RTN Neurons
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Rat brains were immediately removed after endpoint physiological experiments, frozen in liquid nitrogen and stored at −80°C for subsequent use. Frozen brainstem containing RTN neurons were obtained by cutting 600-μm of coronal section through the medulla oblongata between 11.9 to 11.3 mm caudal to bregma using a cryostat. The RTN was punched using a blunt 15‐gauge needle attached to a syringe as previously described.23 (link),24 (link) RNA isolation and cDNA synthesis were performed using the RNAqueous Micro® (cat# AM1931, Ambion) and iScript® (cat# 1725037 Promega) kits respectively, according to manufacturer instructions. RNA purity was assessed by spectrophotometry through the 260/280 ratio (1.88 ± 0.11). Gene expression was assessed by SYBR green chemistry real-time PCR following reverse transcription of total RNA. Real-time PCR was performed using the ABI prism 7700 Sequence Detection System (Applied Biosystems). β-Actin mRNA was quantified as an internal control for each sample and quantifications were performed using the 2-ΔΔCT method.
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