ANALYTICAL
Analytical Biochemistry 323 (2003) 252–255
BIOCHEMISTRY
www.elsevier.com/locate/yabio
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: [email protected] (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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