The stability of the nifH mRNA was determined in A1501 and the Δhfq or Δhfq (pLhfq) strains grown under nitrogen fixation conditions for 6 h followed by the addition of rifampicin (400 μg/mL, final concentration). Aliquots were added to two volumes of RNA protect (Sigma). After incubating at room temperature for 5 min, the samples were centrifuged for 5 min at 4°C and 12,000 rpm, and the pellets were quickly frozen in liquid nitrogen and stored at −80°C until they were ready for use. Total RNA was isolated with an innuPREP RNA Mini Kit (Analytik Jena) according to the manufacturer’s instructions. Full-length cDNAs were synthesized from total RNA using a First Strand cDNA synthesis kit (Takara Bio) and were used to estimate mRNA levels with qRT-PCR. The primers used to measure the mRNA half-life are listed in Table S4 (RT nifH F/RT nifH R). The relative mRNA concentration was calculated by the comparative threshold cycle (2−ΔΔCT) method with 16S rRNA as the endogenous reference. Data are presented as the percentage of nifH transcript levels relative to the amount of these transcripts at time point zero.
Rna protect
Manufactured by Merck Group
Sourced in United States, United Kingdom
RNA protect is a reagent designed to stabilize and protect RNA molecules during sample collection, storage, and transportation. It preserves the integrity of RNA, preventing degradation and enabling reliable downstream analysis.
Automatically generated - may contain errors
5 protocols using rna protect
1
Quantifying nifH mRNA Stability
2
Measuring Nitrogen Fixation Gene mRNA Stability
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To assay nfiR, nifD, and nifK mRNA stability, 2-ml bacterial samples incubated for 5 h under nitrogen fixation conditions were collected at different times (0, 1, 3, 5, and 7 min, or 0, 5, 10, 15, 20, 25, and 30 min) right after the addition of rifampin (400 μg/ml). Two volumes of RNAprotect (Sigma, USA) were added to each sample to prevent RNA degradation, and the samples were immediately frozen in liquid nitrogen. Total RNA was isolated from each sample for use in estimating mRNA levels by qRT-PCR. The primers used for measurement of the half-life of the selected gene mRNAs are listed in Table S2 in the supplemental material, and qRT-PCR was performed as described above. Data are presented as percent mRNA levels relative to time point zero.
3
Isolation of Cerebral Blood Vessels from Mice
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Cerebral blood vessels of mice were isolated according to Miguel Gama-Sosa’s purification protocol, with some adaptations [35 (link)]. Briefly, mice were sacrificed and their hippocampi were immediately dissected and frozen. Hippocampi of each mouse were gently homogenized in 4 ml of cold 18% w/v Dextran solution in PBS, containing the RNase inhibitors: RNA protect (Sigma, #R7397) and RNA secure (ThermoFisher, #AM7006) and the detergent NP-40 (ThermoFisher, #28324). Homogenization was performed using a Teflon pestle tissue homogenizer and overhead stirrer (10 strokes). Homogenization resulted in a thick homogenate of low density that was overlaid over 5 ml of Ficoll (1.077 g\ml) to form a single-step discontinuous gradient. Centrifugation was performed for 30 min at 400 g and 4 °C. Pellets were then resuspended, washed twice with PBS and the final pellet was suspended in 1 ml Trizol for RNA extraction.
4
Laser Microdissection of Tumor Tissues
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Fresh-frozen patient tumors were embedded in optimal cutting temperature (OCT) compound and sectioned (8 µm) onto glass slides for hematoxylin and eosin (H&E) staining for pathology review or onto polyethylene naphthalate (PEN) membrane slides for laser microdissection. All PEN membrane sections were stained with H&E and stains for sections destined for LMD harvests for nucleic acid extraction included RNase inhibitors (RNAProtect, Sigma Aldrich). Tissue sections destined for DNA, RNA, and protein extractions were generated from sequential sections generated from each patient tumor block. Laser microdissection was performed (LMD 6500, Leica Microsystems, Wetzlar, Germany) to harvest cellular populations of interest from pathologically-defined regions. Enrichment of tumor and stroma cell populations was performed to achieve greater than 95% purity for each cellular population, avoiding regions of fat or necrosis.
5
HPRT Mutant Colony RNA Extraction
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Solvent control and 10μM B[a]P treated HPRT mutant colonies were transferred to 24-well plates containing 2ml growth medium per well and grown for 5 days at 37°C, 5% CO 2 , after which 300µl of the individual colony was carefully extracted and mixed with 1.5 ml RNA protect (1:5, Sigma, UK) and stored at -20°C for RNA extraction.
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