ANALYTICAL

Analytical Biochemistry 323 (2003) 252–255

BIOCHEMISTRY

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Notes & Tips

Agarose gel size fractionation of RNA for the cloningof full-length cDNAs

Alan Jackson, Phil-Eric Jiao, Irene Ni, and Glenn K. Fu*

Incyte Corp., 3160 Porter Dr., Palo Alto, CA 94304, USA

Received 19 September 2003

The generation of high-quality full-length cDNA

libraries is of critical importance for large-scale EST1

sequencing and clone collection efforts. The ability of

these programs to characterize full-length expressed

genes through EST datasets and to identify full-length

clone reagents for downstream functional experiments

largely depends on the quality of the cDNA librariesused. Several improvements for full-length cDNA li-

brary construction have been reported. Some are

based on modifications of the reverse transcriptase

enzyme to reduce inherent RNase activity [1,2], while

others include modifications to the reverse transcrip-

tion conditions [3,4] or the cloning method [5–8] to

maximize full-length cDNA synthesis. Several strate-

gies that are based on the selection of full-lengthcDNAs containing the mRNA cap structure have also

been described [9–15]. However, despite the applica-

tion of these strategies for cDNA library construction,

many resulting clones still represent only mRNA

transcript fragments. Consequently, the 50 gene ends

are frequently missing from EST datasets and se-

quenced clones often do not contain the complete

open reading frames necessary for the expression ofthe encoded full-length proteins. Given the importance

of creating cDNA libraries representing full-length

cDNAs, we sought to improve current library con-

struction methods by introducing an RNA size frac-

tionation step prior to cDNA synthesis. We show that

this procedure when used in combination with cDNA

size fractionation can significantly increase the number

of full-length clones in a library.

* Corresponding author. Fax: 1-650-845-4664.

E-mail address: gfu@incyte.com (G.K. Fu).1 Abbreviations used: EST, expressed sequence tag; DMSO,

dimethyl sulfoxide.

0003-2697/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2003.10.007

Materials and methods

RNA isolation and size fractionation. Total RNA was

isolated from Cynomolgus monkey brain tissue using

Trizol (Invitrogen, CA). Messenger RNA was isolated

using Oligotex beads (Qiagen, CA) and treated with

DNase I (Invitrogen). A 10-cm-long by 7-cm-wide 1.4%gel was prepared with SeaPlaque low-melting-point

agarose (Cambrex Corp., NJ) using 1� TAE. The

solidified gel was soaked in 5 vol of 36% DMSO in 1�TAE overnight at 4 �C; 10 lg mRNA sample was heat

denatured for 5min at 70 �C in 50% DMSO and quickly

chilled on ice prior to electrophoresis at 5V/cm in 1�TAE/30% DMSO running buffer. After electrophoresis,

the molecular weights of RNA samples were determinedby cutting out an adjacent lane of RNA molecular

weight marker for staining with ethidium bromide. Each

excised gel piece corresponded to approximately 200 llof agarose. The gel piece was soaked in 1ml of H2O at

room temperature for 15min with gentle agitation. The

H2O was removed, 20 ll of 10� b-agarase buffer (New

England Biolabs, MA) was added, and the tubes were

incubated at 70 �C for 5min to melt the agarose beforeincubation at 42 �C for 20min with 20 units of b-agaraseenzyme (New England Biolabs). The RNA was recov-

ered from liquefied agarose using phenol chloroform

extraction followed by ethanol precipitation in the

presence of NH4OAc and 20 lg glycogen carrier (Invit-

rogen).

cDNA synthesis and plasmid cloning. First-strand

cDNA synthesis was accomplished using 100–200 ng ofmRNA and 100 ng oligo(dT)–NotI primer-adaptor with

an RNase H minus MMLV Reverse Transcriptase

(Promega, WI). Second-strand cDNA was generated

using the strand replacement technique [16]. The cDNA

ends were made blunt with T4 DNA polymerase (Pro-

mega) and ligated to EcoRI–XhoI adaptors (Operon,

Notes & Tips / Analytical Biochemistry 323 (2003) 252–255 253

CA) overnight at 16 �C before digestion with NotI (NewEngland Biolabs) at 37 �C for 3 h. cDNA was then size

selected using agarose gel electrophoresis and cloned

into a modified pSPORT vector (Invitrogen). DNA se-

quencing was performed using MegaBACE DNA Se-

quencers (Amersham Biosciences, NJ). Successful

sequences were compared to gb135pri and genpept135

[17] using BLAST [18].

Results and discussion

The separation of RNA through gel matrix is a

routine procedure performed in many laboratories. To

prevent the formation of secondary structures that will

interfere with electrophoretic migration, RNA must be

denatured in preparation for molecular weight deter-mination by agarose gel electrophoresis. Typically,

RNA is denatured either with 1M glyoxal and 50%

DMSO [19] or by treatment with 2.2M formaldehyde in

the presence of 50% formamide [20]. Denatured RNA

treated with these chemicals maintains the ability to

base-pair with complementary RNA or DNA probes

but loses the ability to serve as suitable templates for

reverse transcription. Urea [21] and DMSO [22] havealso been show to be effective solvents for RNA dena-

turation. However, the thermal decomposition products

of urea, such as ammonia, nitrogen oxides, and cyanuric

and cyanic acids [23], formed during heat treatment of

RNA samples or during electrophoresis can lead to

RNA degradation. In this study, we experimented with

a DMSO–agarose gel system to perform the size frac-

tionation of RNA samples under denaturing conditions.RNA is denatured in 50% DMSO at 70 �C for 5min.

Once denatured, RNA may be separated in agarose gels

that do not contain denaturants such as formaldehyde

without affecting the electrophoretic migration of RNA

molecules [24]. However, to prevent any renaturation of

the RNA molecules during electrophoresis, we used

agarose gels containing 36% DMSO. The use of DMSO

in agarose gel electrophoresis has not been describedbefore, but in one study the replacement of 7M urea

with only 5% DMSO in polyacrylamide gel electro-

phoresis kept single-stranded nucleic acids in a dena-

tured state and enabled the successful DNA sequencing

of almost 900 bp with 98.5% accuracy [25]. Higher

concentrations of DMSO were not used as increased

amounts led to soft agarose gels that were difficult to

handle. From 10 lg of mRNA, we isolated 10 distinctsize fractions. The overall RNA recovery yield for the

size fractionation procedure was 73%, as determined by

spectrophotometric quantitation of the gel-extracted

RNA. In prior experiments using RNA molecular

weight markers as test samples for the RNA size frac-

tionation procedure, we have been able to size frac-

tionate as little as 200 ng of RNA with average recovery

yields of approximately 70–80%. The molecular weightand intactness of each sample were determined by aga-

rose gel electrophoresis (Fig. 1A). The molecular

weights of the double-stranded cDNAs synthesized from

each mRNA size fraction were determined by electro-

phoresis through a 1� TAE agarose gel (Fig. 1B). To-

gether, these figures show that RNA can be efficiently

size fractionated by electrophoresis through gel matrix

containing DMSO as a denaturant and that the frac-tionated RNA samples are suitable templates for cDNA

synthesis. As expected, many cDNAs do not reverse

transcribe to the 50 end of the template RNA due to

poor processivity of the MMLV enzyme and to the

secondary structures present within the template RNAs.

This is most apparent with longer mRNA templates as

very little full-length cDNA could be synthesized (lanes

9 and 10, Fig. 1B). The use of cDNA fractionation toincrease the number of full-length cDNA clones in a

library has been previously described [26,27]. However,

mRNA size fractionation prior to cDNA synthesis has

never been described, perhaps because the recovery of

RNA from denaturing agarose gels post electrophoresis

in a form suitable for reverse transcription has not been

possible. Here, as the molecular weights of the size

fractionated mRNA samples are known, cDNA ofmolecular weights corresponding to the input RNA sizes

can then be isolated and cloned. This ensures that most

of the shorter, non-full-length cDNAs are removed

during cDNA size fractionation. The inclusion of the

RNA size fractionation step is particularly important

for larger mRNAs as the majority of cDNAs synthe-

sized are not full length. Insufficient cDNA was recov-

ered for the largest two mRNA fractions so only eightsuccessful cDNA libraries were constructed. The cDNA

insert size distribution of all clones in each library was

determined by amplification of each library in Esche-

richia coli, followed by agarose gel electrophoresis of the

NotI-linearized plasmid library (Fig. 1C). Our results

show that the cDNA insert lengths of the resulting

clones correspond to the mRNA template lengths used

for reverse transcription and that the cloned cDNAs aretherefore most likely to represent full-length mRNAs.

To determine the contribution of RNA size fraction-

ation toward increasing the number of full-length clones

in a cDNA library, we performed DNA sequencing on

192 randomly selected clones from each library and

from two control cDNA libraries made without the

RNA size fractionation step. Table 1 shows our results

in the identification of full-length cDNAs and themRNA lengths of the sequenced clones as done by

BLAST comparison to GenBank nucleotide and protein

sequence databases. We found that for smaller mRNAs

(under 2 kb), the RNA size fractionation step offers a

modest increase of 10.7% in the number of full-length

clones (46.9% in unsized vs 57.6% in sized, Table 1).

However, with larger mRNAs, a significant increase of

Fig. 1. Molecular weight of mRNA and cDNA samples as determined by agarose gel electrophoresis. (A) Size fractionated mRNA samples were

electrophoresed over a 1.0% denaturing agarose gel stained with ethidium bromide. Lanes 2–11 are size fractionated mRNAs isolated from the

starting mRNA sample in lane 1. (B) Autoradiogram of double-stranded cDNAs synthesized from the size fractionated mRNAs in the presence of

[32P]dCTP. The cDNA samples were electrophoresed on a 1% TAE agarose gel. (C) Determination of cDNA insert lengths of plasmid clones. Each

resultant cDNA library was amplified by transformation and growth in E. coli. Plasmid DNA was isolated, linearized by restriction with NotI, and

electrophoresed in agarose gel stained with ethidium bromide. Nonrecombinant plasmids are as indicated by the arrow.

Table 1

Full-length cDNA clones identified in mRNA size fractionated cDNA libraries

Library mRNA size

fraction (bp)

cDNA size

fraction (bp)

Average cDNA

clone size (bp)

Successful

sequences

Sequences identified

by BLAST

Average mRNA

size (bp)

Percentage full

length

1 800–1000 800–1000 1000 148 85 1793 57.6%

2 1000–1500 1000–1500 1400 131 76 2101 52.6%

3 1500–2000 1500–2000 2000 158 99 2433 54.5%

4 2000–2500 2000–2500 2300 163 99 3160 53.5%

5 2500–3000 2500–3000 3000 166 83 3456 51.8%

6 3000–3500 3000–3500 3300 141 67 3677 38.8%

7 3500–4000 3500–4000 3500 152 56 3636 32.1%

8 4000–4500 4000–4500 4000 131 46 5093 23.9%

Control 1 No fractiona-

tion

500–2000 1300 136 49 1596 46.9%

Control 2 No fractiona-

tion

2000–4000 2200 136 61 2997 36.1%

DNA sequencing was attempted on 192 randomly selected clones from each of the 8 mRNA size fractionated cDNA libraries and from 2 non-size

fractionated control libraries. The average mRNA sizes for successful sequences were identified by BLAST comparison to known mRNA sequences.

A clone was determined to be full length if its cDNA insert contains both a poly(A) stretch and the open reading frame start codon.

254 Notes & Tips / Analytical Biochemistry 323 (2003) 252–255

17.4% in the number of full-length clones was observed

(36.1% in unsized vs 53.5% in sized, Table 1).

Our results clearly show that the inclusion of an

mRNA size fractionation step would be of significant

value toward the preparation of high-quality, full-length

cDNA libraries. We speculate that this RNA size frac-

tionation procedure can be used in conjunction with

other reported methods employing manipulations at the

Notes & Tips / Analytical Biochemistry 323 (2003) 252–255 255

cDNA level to greatly enhance for full-length geneswithin cDNA libraries. The DMSO agarose gel system

described here can also be used for the recovery of un-

modified, undamaged RNA after electrophoresis sepa-

ration for cDNA synthesis, and for other molecular

biology applications requiring intact, unmodified RNA.

Acknowledgments

We thank Laura Stuve and Richard Goold for

helpful comments and the Incyte HTPS group for DNA

sequencing.

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