GAL1-3HA-ERB1 cells containing plasmids expressing wild-type or mutant erb1 protein from the pOBD2 vector were grown to an OD600 of 0.6, washed with PBS and treated with 100 mM 2-methyl nicotinic acid imidazolide (NAI) for 20 min at 30°C. Total RNA was extracted with acid phenol:chloroform:isoamyl alcohol (Ambion). Primer extensions of 5 μg of total RNA extracted from NAI-treated cells were performed using Transcriptor Reverse Transcriptase (Roche Diagnostics) with oligonucleotides designed to base-pair with the ITS2 sequence in 27S pre-rRNA (20 (link)).
Acid phenol chloroform isoamyl alcohol
Manufactured by Thermo Fisher Scientific
Acid phenol:chloroform:isoamyl alcohol is a solution used for the extraction and purification of nucleic acids, such as DNA and RNA, from various biological samples. It is a mixture of phenol, chloroform, and isoamyl alcohol, which facilitates the separation of nucleic acids from other cellular components during the extraction process. The solution aids in the denaturation and removal of proteins, lipids, and other impurities, allowing for the efficient isolation of high-quality nucleic acids for downstream applications.
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Lab products found in correlation
Chloroform, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Neutral phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (2 mentions)
Transcriptor reverse transcriptase, by Roche (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Glass bead, by Merck Group (1 mentions)
Superscript 4 vilo, by Thermo Fisher Scientific (1 mentions)
Dna free kit, by Thermo Fisher Scientific (1 mentions)
Abi 7900 cycler, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Random decamers, by Thermo Fisher Scientific (1 mentions)
Omniscript reverse transcriptase kit, by Qiagen (1 mentions)
Beckman j 6b, by Beckman Coulter (1 mentions)
Oligo dt, by Qiagen (1 mentions)
7 protocols using acid phenol chloroform isoamyl alcohol
1
NAI-Mediated Pre-rRNA Structural Analysis
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GAL1-3HA-ERB1 cells containing plasmids expressing wild-type or mutant erb1 protein from the pOBD2 vector were grown to an OD600 of 0.6, washed with PBS and treated with 100 mM 2-methyl nicotinic acid imidazolide (NAI) for 20 min at 30°C. Total RNA was extracted with acid phenol:chloroform:isoamyl alcohol (Ambion). Primer extensions of 5 μg of total RNA extracted from NAI-treated cells were performed using Transcriptor Reverse Transcriptase (Roche Diagnostics) with oligonucleotides designed to base-pair with the ITS2 sequence in 27S pre-rRNA (20 (link)).
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GAL1-3HA-ERB1 cells containing plasmids expressing wild-type or mutant erb1 protein from the pOBD2 vector were grown to an OD600 of 0.6, washed with PBS and treated with 100 mM 2-methyl nicotinic acid imidazolide (NAI) for 20 min at 30°C. Total RNA was extracted with acid phenol:chloroform:isoamyl alcohol (Ambion). Primer extensions of 5 μg of total RNA extracted from NAI-treated cells were performed using Transcriptor Reverse Transcriptase (Roche Diagnostics) with oligonucleotides designed to base-pair with the ITS2 sequence in 27S pre-rRNA (20 (link)).
2
Quantitative PCR Analysis of unc-51 in C. elegans
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RNA was isolated from young adult animals. Worms were homogenized, and RNA was extracted with Trizol reagent (Invitrogen) using repeated cycles of vortexing (4 °C) with 500- µm glass beads (Sigma) and freeze-thawing in liquid N2. RNA was isolated with chloroform (Sigma) and acid phenol:chloroform:Isoamyl alcohol (125:24:1), pH 4.5 (Ambion). Contaminating DNA was removed by subjecting 10 µg of RNA to DNA-free Kit (Ambion). cDNA was generated from 1 µg of RNA with Superscript IV Vilo (Invitrogen). Quantitative PCR was carried out using Power-Up Sybr Green Master Mix in the ABI 7900 cycler (Applied Biosystems). All qPCR amplifications were performed in triplicate using cDNA and no RT negative controls. Quantitation of unc-51 was normalized to tba-1, pmp-3, and Y45F10D.4 reference genes75 (link) using the comparative C(T) method for analysis76 (link). All primers used for qPCR are listed in Table S5 .
3
RNA Photoaffinity Labeling Protocol
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5 μg total RNA (isolated from HeLa cells) or 10 pmol of in vitro transcribed RNA in 6 μl metal-free water was heated for 2 min at 95°C. The RNA was then flash cooled on ice for 1 min, and brought to room temperature. 3 μl of 3× RNA folding buffer (333 mM HEPES, pH 8.0, 20 mM MgCl2 and 333 mM NaCl) was added, and the RNA was allowed to equilibrate at 37°C for 5 min. To this mixture 1 μl of 3 M NAz, 1 M NAz-N3 in DMSO (+) or DMSO (−) was added. Reactions were then exposed to 20 W lamp (Zilla Desert UVB 50) UV light for 3 min for NAz, and 10 minutes for NAz-N3. Reactions were brought up to 200 μl water, and extracted once with 200 μl acid phenol/chloroform/isoamyl alcohol (pH 4.5, Ambion), and washed twice with 200 μl chloroform (Sigma). Samples were precipitated by adding 20 μl 3 M NaOAc (pH 5.2), 1 μl glycoblue (20 μg/μl) and 600 μl EtOH. Pellets were washed twice with 70% cold ethanol and resuspended in 5 μl nuclease free water.
4
Intranasal Bacterial Infection in Mice
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Animal experiments were approved by the University of Adelaide Animal Ethics Committee. Groups of 12 outbred 5–6-week-old female Swiss (CD-1) mice (48 in total), were anesthetized by intraperitoneal injection of pentobarbital sodium (Nembutal) and challenged intranasally (IN) with 50 µl of bacterial suspension containing approximately 1 × 108 CFU in SB of 4559-Blood, 9–47-Ear, 4559M or 9–47M. The challenge dose was confirmed retrospectively by serial dilution and plating on BA. Mice were euthanized by CO2 asphyxiation at 6 h and lungs placed in 1 ml TRIzol (Thermo Fisher). RNA was then extracted using acid-phenol-chloroform-isoamyl alcohol (125:21:1; pH 4.5; Ambion) and purified using the RNeasy minikit (Qiagen). For subsequent dual RNA-seq analyses, there were three replicates per strain, with each replicate derived from the lungs of four mice.
5
Milk RNA Isolation and Analysis Protocol
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Milk samples were collected from the morning milking on the final day of each period and subjected to RNA analysis using the methods of Brenaut et al. (2012) (link) and Cánovas et al. (2014) (link) with the following modifications. Milk samples (45 mL) were collected into RNase-free sample bottles, transferred into sterile 50 mL conical tubes, and stored on ice. Samples were centrifuged at 2,000 × g for 10 min at 4°C (Beckman J-6B; Beckman Coulter, Inc., Indianapolis, IN) to separate fat from other milk components. The resultant fat was suspended in RNase-free water (1:1), and an equal volume of TRIzol LS (Life Technologies, Grand Island, NY) was added and thoroughly mixed. All samples were stored at -80°C until analysis. Chloroform was added to the thawed samples, they were centrifuged at 4°C and 12,000 × g for 15 min, and the resultant aqueous phase was combined with an equal volume of acid phenol: chloroform-isoamyl alcohol (Ambion, Life Technologies). Samples were centrifuged at 12,000 × g at 4°C for 10 min, and the aqueous phase was combined with an equal volume of 70% ethanol, vortexed, and purified using the RNeasy Mini Kit and treated with DNase I (Qiagen Inc.). A 2 μg aliquot of the resulting RNA was reverse-transcribed using an Omniscript reverse transcriptase kit (Qiagen Inc.), oligo-dT (0.05 μg/μL; Qiagen Inc.), and random decamers (10 μM; Ambion).
6
Total RNA Extraction from Bacterial Cultures
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Total RNA was extracted from exponential phase cultures growing either in LB or in MOPS EZ rich defined medium using hot phenol lysis method described previously (Massé et al. 2003 (link)). Briefly 700 µL of samples were removed from growing cultures and added to a mixture containing 800 µL of acid phenol–chloroform-isoamyl alcohol (pH of 4.3; Fisher Scientific) and 100 µL of lysis buffer (320 mM sodium acetate [pH 4.6], 8% SDS, 16 mM EDTA) equilibrated to 65°C. Samples were mixed at 65°C for 5 min and centrifuged for 30 min at 4°C to separate phases. The upper aqueous phase was extracted a second time with equal volume of neutral phenol–chloroform-isoamyl alcohol (pH of 6.7; Fisher Scientific). RNA was alcohol-precipitated and resuspended in DEPC-treated water. RNA concentration was measured using Nano Drop 2000 (Thermo Fisher Scientific).
7
RNA Stability Determination Protocol
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To determine RNA stabilities, cultures were grown in BHI to exponential phase (OD620 ≈ 0.15) as described above, and a culture sample (0 h after the end of log-phase growth [T0]) was collected. Rifampin was added to inhibit transcription, and additional samples were collected 5, 10, 20, and 30 min after rifampin addition. All samples were subjected to hot phenol lysis as described previously (80 (link)). Briefly, 700 μl of sample was added to a mixture containing 800 μl of acid phenol–chloroform-isoamyl alcohol, pH 4.3 (Fisher Scientific), and 100 μl of lysis buffer (320 mM sodium acetate [pH 4.6], 8% [wt/vol] SDS, and 16 mM EDTA) equilibrated to 65°C. Samples were mixed at 65°C for 5 min and centrifuged for 30 min at 4°C to separate phases. The upper aqueous phase was extracted a second time with an equal volume of neutral phenol–chloroform-isoamyl alcohol, pH 6.7 (Fisher Scientific). RNA was ethanol precipitated and resuspended in DEPC-treated water. RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific).
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