TRIzol Reagent
RNA isolation / purification Tissue - Human Fallopian tubes
Protocol tips
| Protocol tips |
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| - For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it. |
Publication protocol
total RNA was isolated from the Fallopian tube tissue using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions, followed by DNase treatment. The purity and quality of the RNA were assessed spectrophotometrically by determining their absorbance ratio A260/A280 and electrophoresis of samples on 1.5% denaturing agarose gels. First and second-strand cDNA were prepared according to the Affymetrix microarray preparation protocol. Biotinylated-cRNA was then prepared using a BioArray High Yield T7 Transcript labelling kit (Enzo, Farmingdale, NY, USA) and chemically fragmented. Biotinylated cRNAs were hybridized overnight to a high-density Affimetrix Human Genome U133 Plus 2.0 oligonucleotide microarray genechip containing 54,600 probe sets (42,203 annotated genes and 12,397 expressed sequence tags (EST)) at the Stanford University School of Medicine Protein and Nucleic Acid Facility. Subsequently, fluorescent labelling and laser confocal scanning with an HR 3000 scanner was conducted. Generated data were extracted for analysis using the Affymetrix Gene-Chip Operating Software (GCOS) version 1.1.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
5 years ago
Author:
Paul G. Macon
RNA isolation from tissue
How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?
Discussion
5 years ago
Author:
Aaron Stege
Problem in phase separation after using serum/plasma kit
I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?
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Manufacturer protocol
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