About 2 x 106 cells per sample were harvested in 1.5 ml Trizol (Life Technologies GmbH, Darmstadt, Germany), homogenized and mixed with 0.45 ml chloroform. Phases were separated by centrifugation. For RNA precipitation the upper aqueous phase was aspirated and 1.25 ml isopropanol were added, mixed and centrifuged. Subsequently the pellet was washed with 75% ethanol, then dried and finally dissolved in water. The total RNA concentration of individual samples was determined photometrically using the NanoDrop 1000 ND-1000 (Peqlab, Erlangen, Germany). Overall RNA quality was assessed by gel electrophoresis using a 1% agarose gel with a 1 KB molecular weight marker separated in parallel.
Nanodrop 1000 nd 1000
Manufactured by Avantor
Sourced in Germany
The NanoDrop 1000 ND-1000 is a spectrophotometer designed for measuring the concentration and purity of nucleic acid and protein samples. It utilizes a patented sample-retention technology that requires only 1-2 microliters of sample for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Lightcycler dna master sybr green 1, by Roche (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Goscript reverse transcription system kit, by Promega (1 mentions)
4 protocols using nanodrop 1000 nd 1000
1
RNA Isolation from Cell Samples
2
RNA Extraction from Cultured Cells
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The cells were washed with PBS and lysed in Trizol (Invitrogen, Life Technologies GmbH) to harvest approximately 2 × 106 cells per 1.5 mL Trizol. The samples were homogenized by vigorous shaking. A total of 0.3 mL chloroform was added per 1 mL Trizol, and the samples were mixed for 15 s by vigorous shaking. Phase separation was allowed by placing the samples on the bench top for 10 min followed by centrifugation at 12,000 ×g for 25 min at 4°C. The upper aqueous phase was transferred to a fresh tube. A total of 0.75 mL isopropanol per 1 mL Trizol was added, thoroughly mixed, and incubated for 10 min and centrifuged at 12,000 ×g at 4°C to precipitate the RNA. The RNA pellet was washed with 0.5 mL ethanol per 1 mL Trizol and centrifugation at 12,000 ×g for 10 min at 4°C. The supernatant was aspirated and the sediment was air dried for 15 min. Total RNA was dissolved in nuclease-free water and photometrically quantified by NanoDrop 1000 ND-1000 (Peqlab, Erlangen, Germany) measurement. Moreover, approximately 250 ng RNA were analyzed on 1% Agarose gel with a 1-KB marker for overall RNA quality control.
3
Efficient miRNA Profiling from Cellular RNA
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For miRNA profiling, about 2 × 106 cells were harvested in 1.5 ml Trizol (Life Technologies), homogenized, mixed with 0.45 ml chloroform and phase separated by centrifugation. RNA was precipitated by aspiration of the upper aqueous phase, addition of 1.25 ml isopropanol, mixing and centrifugation. The pellet was washed with 75% ethanol, then dried and dissolved in water. Total RNA concentrations were determined photometrically using the NanoDrop 1000 ND-1000 (Peqlab, Erlangen, Germany). Overall RNA quality was assessed on a 1% agarose gel with a 1 KB molecular weight marker separated in parallel. Cells from MEK-inhibitor experiments were lyzed with 700 μl Qiazol (Qiagen, Hilden, Germany), thoroughly mixed with 140 μl chloroform and centrifuged at 12,000 × g at 4°C for 15 minutes. The upper aqueous phase was mixed with 1.5 volumes of ethanol and loaded onto an RNeasy Mini spin column (RNeasy Mini Kit, Qiagen). RNA extraction was performed according to the manufacturer’s instructions.
4
RNA Isolation and Gene Expression Analysis Protocol
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Total RNA isolation and gene expression analysis were conducted as previously described [30 (link),33 (link)]. The total RNA from endometrial and conceptus tissue samples was isolated using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA). The quality of the RNA was assessed using Agilent 2100 Bioanalyzer (Agilent Technology, Palo Alto, CA, USA). The RNA integrity numbers (RIN) of the samples were at least 7. The RNA amount was spectrophotometrically determined at 260 nm by a Nanodrop 1000 ND-1000 (peqLab Biotechnologie GmbH). GoScript™ Reverse Transcription System kit was used to synthesise complementary DNA (cDNA) as per manufactures instructions (Promega, Switzerland) and the quantitative PCR reactions were carried out using the LightCycler DNA Master SYBR Green I protocol (Roche Diagnostics). The gene expression of the target genes was normalised against the reference gene polyubiquitin (UBQ3) to generate a relative RNA expression (ΔCq). To avoid negative values while permitting an evaluation of a relative comparison between two genes, the resulting ΔCq value was subtracted from an arbitrary value of 20. Thus, high ΔCq values represent high transcript abundances. The primer pairs for all the genes analysed are presented in Table 2 .
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