| | Re: wants to work on microRNA assay in human prostate cancer
Micro-RNA (miRNA) Analysis of Prostate Tumor Samples : miRNA is obtained from cell or tissue samples using a two-step process : A) Total RNAs isolation, B) miRNAs extraction from total RNAs :
1) Tumor and normal adjacent tissue (NAT) samples were obtained from prostate cancer patients
2) Total RNA from these samples are isolated, following the steps below described :
a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about 1 M guanidinium is produced : 1) for tissue samples, 1 ml of Lysis/Binding Solution (4 M Guanidinium Thiocyanate, 0.5% N Lauryl Sarcosine, 25 mM Na-cltrate pH 7.2, 0.1 M 2-Mercaptoethanol) per 0.1 g of tissue is added. The samples were kept cold, and the tissue is thoroughly disrupted in Lysis/Binding Solution using a motorized rotor-stator homogenizer; b) for cell samples, 0.6 ml of Lysis/Binding Solution is added per 107 cells. The cells were lysed by vortexing for 30-60 sec. A 1/10 volume of glacial acetic acid is added to the homogenate. For example, if the lysate volume is 300 µl, 30 µl glacial acetic acid is added. The sample is then mixed well by vortexing or inverting the tube several times before leaving it on ice for 10 min.
b) extracting miRNA molecules from the lysate with an extraction solution comprising phenol : A volume of extracting solution (acid-Phenol [phenol equilibrated with 0.1 M citrate buffer at pH 4.3 ± 0.2]:Chloroform:isoamylalcohol mixture of isomers [IAA – Fluka cod. 59100] with ratio 125:24:1 [v/v], stored in 50 ml or less in capped amber or foil wrapped bottles to avoid oxidation and breakdown product at 4 °C [stable 6 months if not opened frequently] or -20 °C [stable 1 year or more]) equal to the sample lysate volume is added to the sample and then vortexed for 30-60 sec to mix it. The mixture is centrifuged for 5 min at maximum speed (10,000 g about 10900 rpm) at room temperature to separate the aqueous and organic phases. The aqueous (upper) phase is carefully removed without disturbing the lower phase and transferred to a fresh tube.
c) adding to the lysate an alcohol solution for form a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70% : 1.25 volumes of 100% ethanol is added to the aqueous phase (e.g., if 300 µl is recovered from the aqueous phase, 375 µl of ethanol is added). The lysate/ethanol mixture is pipetted onto a commercial or home-made glass fiber filter column. The column is then centrifuged at 10,000 g for approximately 15 sec. to pass the mixture through the filter. After discarding the flow-through, the process is repeated until all of the lysate/ethanol mixture passed through the filter. 700 µl of miRNA Wash Solution A (1.6 M Guanidinium Thiocyanate, 30% EtOH) are applied to the filter column, which is subsequently centrifuged for about 5-10 sec or placed under a vacuum to pull the solution through the filter. The flow-through is discarded. 500 µl of Wash Solution B (100 mM NaCl, 4.5 mM EDTA, 10 mM Tris pH 7.5) is applied to the filter column and drawn through it as in the previous step. The wash is repeated with a second 500 µl aliquot of Wash Solution B. After discarding the flow-through from the last wash, the assembly is spun for 1 min to remove residual fluid from the filter. 100 µl of room temperature nuclease-free water is added to the center of the filter, and the cap is closed. It is spun for approximately 20-30 sec. at maximum speed to recover the total RNA (miRNA, mRNAs and rRNAs). To the isolated total RNA, 300 µl of Lysis/Binding Solution is added. 100 µl of 100% ethanol is then added. It’s mixed thoroughly by vortexing or inverting the tube several times. The lysate/ethanol mixture is pipetted onto a glass fiber filter column and centrifuged for about 15 sec at 10,000 g. The filtrate is collected and a 2/3 volume of 100% ethanol is added to the filtrate (i.e., flow-through) and it’s mixed thoroughly. The filtrate/ethanol mixture (from the previous step) is pipetted onto a new glass fiber filter column. The column is centrifuged at 10,000 g for approximately 15 sec. to pass the mixture through the filter. 700 µl of miRNA Wash Solution are applied to the filter column, which was subsequently centrifuged for about 5-10 sec. or placed under a vacuum to pull the solution through the filter. The flow-through is discarded. 500 µl of Wash Solution B are applied to the filter column and drawn through it as in the previous step. The wash is repeated with a second 500 µl aliquot of Wash Solution B. After discarding the flow-through from the last wash, the assembly is spun for 1 min to remove residual fluid from the filter. 100 µl of room temperature nuclease-free water are added to the center of the filter, and the cap is closed. It’s spun for approximately 20-30 seconds at maximum speed to recover the miRNA. The miRNA is lyophilized and stored at -20°C or lower until labeled for analysis.