Isolation of Size-Specific RNA from Total RNA Using GELase™ Agarose Gel-Digesting Preparation, EPICENTRE® Biotechnologies

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|Isolation of Size-Specific RNA from Total RNA Using GELase™ Agarose Gel-Digesting Preparation|

Construction of cDNA libraries is a time-consuming and labor-intensive process. The desired cDNA clones are often derived from specific transcripts or families of transcripts isolated from total cellular RNA. Libraries constructed from total cellular RNA are often difficult to work with due to high background. Construction of cDNA libraries from size-selected RNA increases the likelihood of identifying full-length clones and decreases the number of background clones in the library, thereby reducing screening time. Here we describe a simple procedure for purifying RNA of defined sizes from total cellular RNA using GELase™ Agarose Gel-Digesting Preparation.

Total yeast RNA was isolated as previously described.¹ Ten micrograms were electrophoresed on a 1.2% LMP-agarose gel containing formaldehyde and then stained with ethidium bromide (Figure 1). The 1-2 kb RNA was excised from the gel and weighed in a tared 15 ml tube. The total weight of the gel slice was 1.3 grams. The RNA was purified according to the GELase protocol shown in Table 1. This protocol is used for nucleic acid purification from gels containing denaturants. Three units of GELase Preparation were used and the digestion was performed for 1 hour.

Figure 1. Ten µg of total yeast RNA electrophoresed on a 1.2% LMP-agarose formaldehyde gel (Lane 2). Lane 1, molecular weight marker.

Figure 2. Results of RNA isolation using GELase Preparation. RNA from 1 to 2 kb in size was isolated from total yeast RNA using GELase Preparation as described and run on a 1% agarose formaldehyde gel. Lane 1, molecular weight marker. Lane 2, RNA from 1 to 2 kb in length isolated from total cellular RNA.

Table 1. Protocol for the Isolation of RNA
from LMP-Agarose Formaldehyde Gels.

GELase Digestion

Cut out the desired region from the gel.

Weigh the gel slice in a tared tube.

Soak the gel slice in three volumes of 1X GELase Buffer for one hour, replacing the buffer with three volumes of fresh buffer at 20 and 40 minutes.

Carefully remove the excess GELase Buffer with a pipette.

Melt the gel slice completely at 70°C.

Equilibrate the molten agarose to 45°C.

Add the appropriate amount of GELase Preparation (1 unit per 600 mg of 1% LMP agarose gel).

Incubate for one hour at 45°C.

Precipitate the RNA as follows.

Ethanol Precipitation

Add one volume of fresh 5M NH₄OAc, pH 7.0. NH₄OAc solutions should be filter-sterilized, not autoclaved.

Add two volumes (4X original volume) of room temperature ethanol. Use of cold ethanol can precipitate the small oligosaccharides that result from GELase digestion.

Pellet the nucleic acid by centrifugation for 30 minutes at room temperature. Do not use a refrigerated centrifuge.

Carefully remove the supernatant with a pipette.

Wash the nucleic acid pellet gently with 70% ethanol.

Centrifuge briefly and carefully remove the ethanol wash with a pipette.

Resuspend in T₁₀E₁ (10 mM Tris, pH 7.5, 1 mM EDTA) and check recovery by agarose gel electrophoresis.

After precipitation, the RNA was resuspended in 100 µl of water and 5 µl were run on a 1% agarose gel containing formaldehyde (Figure 2). RNA from approximately 1 kb to 2 kb in size is present on the gel. This RNA is now ready for cDNA synthesis. (Note: RNA slightly smaller than 1 kb present on the gel is due to differences between the ability of regular agarose and LMP-agarose to resolve nucleic acids.)

Here we have shown the ability of GELase Agarose Gel-Digesting Preparation to purify RNA of defined size. Constructing cDNA libraries from size-selected RNA may reduce the time required to screen the library by reducing the number of background clones.

Rose, Winston and Hieter (1990) Methods in Yeast Genetics, A Laboratory Course Manual. Cold Spring Harbor Press, Cold Spring Harbor, New York, 140.