Formulations for nucleic acid stabilization on solid substrates
a solid substrate and nucleic acid technology, applied in the field of dry solid substrates, can solve the problems of preventing the collection of rna from samples (e.g., biological samples) without significant sample processing, and preserving rna in liquid state for days or weeks at room temperature,
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example 1
General RNA Analysis
[0041]A cultured human lymphocyte cell line (i.e., Jurkat cells) was utilized as the source of total cellular RNA. Cells were dried on 7-mm cellulose discs impregnated with the indicated reagents, stored at room temperature for 10 days in a desiccator cabinet, and cellular nucleic acids were electroeluted in accordance with standard protocols. Briefly, discs were re-hydrated with 15 μL of 2 mg / mL proteinase K in nuclease-free water to remove excess protein and dried for ˜30 min. Punches were placed into individual wells of a 1% Tris-borate-EDTA (TBE) agarose gel and suspended inlX Gel Loading Buffer II containing formamide (Ambion). Cellular nucleic acids were electrophoresed at 110 volts for 1-2 hours, and RNA and DNA were post-stained with SYBR Gold (Invitrogen) and detected using a Typhoon Imager (GE Healthcare). All equipment and surfaces were treated with RNAZap (Ambion) to preserve the integrity of cellular RNA during and subsequent to electro-elution from ...
example 2
Empirical Determination of Favorable Conditions for RNA Extraction and Storage
[0045]The primary purpose of this example was to evaluate the effect of each single factor and the effect of the combination of factors tested (e.g., chelating agent, buffer, pH, protein denaturant, reducing agent, and peptide RNase inhibitor) on preserving RNA on cellulose paper. An additional aspect of this example was to evaluate the presence of reducing agent (DTT) to potentially enhance the effect of the protein denaturant.
[0046]Jurkat cells were again utilized as the source of total cellular RNA, and the cells were applied directly onto cellulose paper samples and air-dried to mimic a typical end-user application. Total cellular RNA was recovered by electroelution, following the protocol described above in Example 1, into a 1% agarose gel and analyzed for 28s:18s rRNA content based on known standards. Samples containing the components listed under each bar on the graph of FIG. 2 were stored for 10 da...
example 3
Continued Empirical Determination of Favorable Conditions for RNA Extraction and Storage
[0048]After key components for storing RNA were identified in Example 2, Example 3 was designed to investigate the effect of DTT and SDS either alone or in combination on the ability to preserve RNA, and the effect of a free radical trap and chelating agent on the performance of GITC / DTT combinations that exhibited favorable RNA stabilization properties in Example 2.
[0049]Jurkat cells were applied directly onto cellulose paper samples and air-dried to mimic a typical end-user application. Total cellular RNA was recovered by electroelution, following the protocol described above in Example 1, into a 1% agarose gel and analyzed for 28s:18s rRNA content based on known standards.
[0050]Cellulose samples were stored for 13 days at room temperature in a desiccator cabinet prior to analysis. Numbers above each bar correspond to the ratio of 28s to 18s rRNA. A 28s:18s ratio >1 generally indicates intact R...
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