Methods and compositions for the rapid isolation of small RNA molecules

Abstract

The present invention provides extraction compositions and methods for the rapid and efficient isolation of small RNA molecules from a biological sample. In particular, the extraction compositions, when contacted with a biological sample, releases the small RNA molecules from the other molecules in a biological sample, and the released small RNA molecules may then be isolated.

Description

FIELD OF THE INVENTION [0001] The present invention relates to methods, compositions, and kits to isolate small RNA molecules from biological samples. BACKGROUND OF THE INVENTION [0002] More than a decade ago a non-coding 22-nucleotide (nt) RNA (lin-4) was discovered that played an important role in the developmental timing of Caenorhabditis elegans. It was not realized, however, until just a just few years ago that small RNA molecules such as lin-4 are ubiquitous and play important regulatory roles in virtually all eukaryotes. Recent work has shown that prokaryotes and viruses also express small regulatory RNA molecules. Thus, in addition to large RNA molecules, such as messenger RNA (mRNA) and ribosomal RNA (rRNA), cells express an array of small RNA molecules, including 5.8S rRNA, 5S rRNA, transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA); micro RNA (miRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), ...

AI Technical Summary

Benefits of technology

[0048] Nine basal solutions were prepared that each comprised 7 M guanidine hydrochloride, 60 mM trizma acetate, and 2% Tween 20, but each had a different pH through titration with acetic acid or NaOH. The pH values were 3.2, 3.4, 3.6, 3.8, 4.0, 5.0, 6.0, 7.0, and 8.0. Nine lysis solutions were prepared by combining each of the nine basal solutions with a 12 M LiCl solution in a 7:3 ratio. The resulting lysis solutions each comprised 4.9 M guanidine hydrochloride, 3.6 M LiCl, 42 mM trizma acetate, and 1.4% Tween 20, and each solution had a different pH. Each lysis solution was further supplemented with 2-mercaptoethanol at 1%.

[0049] Grape leaves were ground to a fine powder in liquid nitrogen and nine 100-mg aliquots were prepared from the powdered material. Each aliquot was lysed in 750 μl of a lysis solution at 55° C. for 4 minutes. The samples were then centrifuged for 5 minutes performed. The supernatant fraction was filtered through a filtration column (C 6866, Sigma-Aldrich, St. Louis, Mo.) by 1 minute of centrifugation to remove carry-over particulates. The clarified lysate was mixed with 830 μl of 100% ethanol and applied to a silica filter binding column (C6991, Sigma-Aldrich, St. Louis, Mo.) in two loadings, with 30 seconds of centrifugation after each loading. The column washed in succession with 500 μl of 100% ethanol, 500 μl of 12 M LiCl, and twice with 500 μl of an alcohol wash solution comprising 80% ethanol and 10 mM tris (pH 7.0). Each wash step was carried out with a short centrifugation (30 seconds or 1 minute). The column was dried by 1 minute of centrifugation and the bound nucleic acids were eluted in 50 μl of RNase-free water and 1 minute of centrifugation. All centrifugation steps were performed in a bench-top microcentrifuge at top speed (14,000×g) at room temperature. The samples were an...

Problems solved by technology

The long delay to the realization of the existence and importance of small RNA could, in part, be attributed to the fact that small RNA molecules are often unintentionally eliminated because of their small sizes from preparations of natural RNA populations.

Furthermore, small RNA molecules represent a very small fraction in terms of weight of the total RNA population, and without removal of abundant RNAs and enrichment of small RNAs, their detection could be severely hampered.

Alcohol precipitation, however, does not quantitatively recover small RNA molecules.

Small RNA, however, binds poorly to the support matrix under the conditions routinely used.

Thus, most existing RNA preparation methods and commercial RNA purification kits are deficient in capturing small RNA.

A major drawback of the current methodology is the use of phenol and chloroform, not only because they pose potential health hazards but also because they are ineffective with certain biological material, such as plant tissues that are rich in phenolic or polyphenolic compounds.

Another drawback of the current methodology is that phase separation and alcohol fractionation are laborious and time consuming, making them incompatible with high throughput and automation demands.