RNA-free animal serum
A serum and animal technology, applied in the field of removing RNA and fetal bovine serum, can solve the problems of unresearched, hindered and contaminated cultures of the influence of culture
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Embodiment 1
[0095] RNA removal from SFB
[0096] Starting with a regular SFB commercial product, an initial determination of total RNA concentration was performed by extracting it with a Trizol product as recommended by the manufacturer and found to be 35 ng / mL.
[0097] The SFB was heated at 56°C for 35 minutes and slowly cooled to room temperature until 20°C was reached. Subsequently, the serum was alkalized using 12N anhydrous NaOH until pH 12 was reached and held in this manner for 15 minutes, then the pH of the alkalized serum was lowered using 1N HCl until physiological pH 7.4 was reached.
[0098] After applying this method, the total RNA concentration was determined by the above-mentioned Trizol extraction method, and the concentration below the detection limit was found by fluorescence spectrometry.
Embodiment 2
[0100] Removal of RNA from SFB in combination with methods of the invention and ultracentrifugation
[0101] Starting with a regular SFB commercial product, which was found to have an RNA concentration of 40 ng / mL, the serum was ultracentrifuged at 100,000 xg for 7 hours at 4°C. At the end of the ultracentrifugation, transfer the sample supernatant to a new sterile container without disturbing the bottom results. To maintain sterile conditions, the serum supernatant was passed through a sterile filter with a pore size of 0.2 microns.
[0102]Subsequently, the SFB supernatant was heated at 56 °C for 35 min and allowed to cool slowly to room temperature until reaching 20 °C; then, the serum was alkalized with anhydrous NaOH until pH 12 was reached and maintained for 15 min, after which 1 N was used HCl lowers the pH of alkalizing serum until physiological pH 7.4 is reached.
[0103] After applying this method, the total RNA concentration was determined by the above-mentioned T...
Embodiment 3
[0105] Removal of RNA from SFB using the method of the present invention in different iterations
[0106] The different serum panels are expected to contain higher amounts of RNA than the other sera. In the case of serum samples with high RNA content, the method of the present invention can be repeated to yield an additive result of its efficiency in RNA removal.
[0107] Starting from serum with RNA content above 45 ng / mL, heat at 56 °C for 35 min and allow to cool slowly to room temperature until reaching 20 °C; then, basify serum with anhydrous NaOH until pH 12 is reached and hold for 15 min , after which the pH of the alkalizing serum was lowered using 1N HCl until a physiological pH of 7.4 was reached.
[0108] Subsequently, the heating, cooling, alkalization and neutralization processes were repeated under the same conditions.
[0109] After two consecutive applications of the method, the total RNA concentration was determined by the Trizol extraction method described ...
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