Nielsen H. RNA: Methods and Protocols

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114 Yeku and Frohman

3. Incubate the reaction for 15 min at 65

◦

C to inactivate SAP,

then spin brieﬂy in a microcentrifuge. At this point, the

products can be stored at –80

◦

4. To visually conﬁrm that the RNA remained intact during

the dephosphorylation step, 2 μg(2μL) of RNA should

be analyzed by electrophoresis through a 1% agarose gel

(TAE buffer) adjacent to a lane containing 2 μgofthe

original RNA preparation. Upon ethidium bromide stain-

ing, degraded RNA will be present as a low molecular weight

smear on the gel rather than a discrete band of high molecu-

lar weight. If evidence of RNA degradation is observed, then

fresh reagents should be prepared and the step repeated.

3.2. Decapping of

Intact RNA

1. Combine and mix the reagents listed in Table 8.2 in a sterile

microfuge tube. Note that the quantity of TAP proposed is

sufﬁcient for the reaction.

2. Incubate the reaction for 1 h at 37

◦

C; then add 200 μLTE

buffer to stop the reaction.

3. Spin columns provided in any RNA extraction kit or from

varied companies can be used for separation of the RNA

from the reaction components. Resuspend the RNA in 40

μLH

O. It is permissible to store the products at this point

at –80

◦

4. 2 μg of RNA should be analyzed by electrophoresis

(through a 1% agarose gel in TAE buffer) adjacent to a

lane containing 2 μg of the original RNA preparation; stain

the gel with ethidium bromide and visually conﬁrm that

the RNA remained intact during the decapping step. A low

molecular weight smear on the gel indicates the presence of

RNA fragments, whereas a discrete band of high molecular

weight corresponds to the desired product.

Table 8.2

Decapping of intact RNA

Component Amount Final

RNA (obtained in Section 3.1, Step 4)48μg48μg

TAP buffer (10×)5μL1×

DTT (0.1 M) 0.5 μL1mM

RNasin (40 U/μL) 1.25 μL50U

TAP (5 U/μL) 1 μL5U

Oto50μL–

Rapid Ampliﬁcation of cDNA Ends (RACE) 115

3.3. Preparation of

RNA Oligonucleotide

1. Using digestion by appropriate restriction enzymes and

buffers, linearize 25 μg of the plasmid to be transcribed.

Ensure that the plasmid is reasonably RNase-free.

2. Eliminate any residual RNase activity in the reaction by treat-

ing the digestion for 30 min at 37

◦

C with 50 μg/mL pro-

teinase K. Extract twice with phenol–chloroform and once

with chlor oform, and collect the DNA by standard ethanol

precipitation.

3. Redissolve the template DNA in 25 μL TE buffer, pH 8.0.

This yields a ﬁnal concentration of approximately 1 μg/μL.

4. Mix the transcription reagents in the order listed in

Table 8.3 in a sterile microfuge tube at room temperature

(25

◦

C).

5. Incubate for 1 h at 37

◦

C to allow transcription to occur.

6. Remove the DNA template by adding 0.5 Kunitz units of

DNase I (RNase-free) for every 20 μL of reaction volume

and incubate for 10 min at 37

◦

7. Analyze 5 μL of the test or preparative reaction by elec-

trophoresis through a 1% agarose gel (TAE buffer). Expect

to see a diffuse band of about the right size for the expected

product (or a bit smaller) in addition to some smearing up

and down the gel.

8. Purify the oligonucleotide by extracting the reaction mix-

ture with phenol–chloroform and then chloroform (keeping

Table 8.3

Preparation of RNA oligonucleotide

Component

Amount (test

scale) Final

Amount (prepar-

ative scale) Final

DEPC-treated H

O4μL– 80μL–

Transcription buffer (5×)2μL1× 40 μL1×

DTT (0.1 M) 1 μL10mM20μL10mM

rUTP (10 mM) 0.5 μL 0.5 mM 10 μL0.5mM

rATP (10 mM) 0.5 μL 0.5 mM 10 μL0.5mM

rCTP (10 mM) 0.5 μL 0.5 mM 10 μL0.5mM

Rgtp (10 mM) 0.5 μL 0.5 mM 10 μL0.5mM

Linearized DNA from

Section 3.3 Step 3

(1 μg/μL)

0.5 μL0.5μg10μL 10.0 μg

RNasin (40 U/μL) 0.25 μL10U5μL 200 U

RNA polymerase (20 U/μL) 0.25 μL5U 5μL 100 U

Total 10 μL – 200 μL–

116 Yeku and Frohman

the aqueous phase each time). Perform buffer exchange and

clean-up of the RNA oligonucleotide by diluting it to 1 mL

with H

O and then concentrating it using a Microcon spin

ﬁlter (pre-rinse the spin ﬁlter with H

O before addition of

sample). Rinse twice more with 1 mL of H

O as above. Note

that Microcon 10 spin ﬁlters have a cutoff size of 20 nt and

Microcon 30 spin ﬁlters have a cutoff size of 60 nt. Be sure

to use Microcon 10 ﬁlters for oligonucleotides smaller than

100 nt, and Microcon 30 spin ﬁlters for anything larger.

9. To check for the concentration and integrity of the sam-

ple, analyze a second aliquot of the oligonucleotide by

electrophoresis thr ough a 1% agarose gel in TAE buffer.

The oligonucleotide distribution pattern should look like

that in Step 7 above. A much smaller band likely indicates

that degradation has occurred and the procedure should be

repeated with fresh material. At this juncture, the product

can be stored at –80

◦

C indeﬁnitely.

3.4. RNA

Oligonucleotide–

Cellular RNA

Ligation

1. Set up two sterile microfuge tubes, one with TAP-tr eated

cellular RNA and another with untreated cellular RNA, and

add the remaining components for the ligation reaction (see

Table 8.4). The tube with the untreated cellular RNA serves

as a negative control.

2. Incubate for 16 h at 17

◦

C. Overnight is permissible.

3. Purify and concentrate the ligation product by spin ﬁltra-

tion with a Microcon 100 spin ﬁlter (rinsing product thr ee

times with RNase-free H

O; pre-rinse ﬁlter with H

O). The

recovered volume after the Micr ocon ﬁltration should not

exceed 20 μL. Products can be stored at –80

◦

4. Analyze one third of the product by electrophoresis through

a 1% agarose gel (TAE buffer) to verify the integrity of the

Table 8.4

RNA oligonucleotide–cellular RNA ligation

Component Amount Final

Ligation buffer (10×)3μL1×

RNasin (40 U/μL) 0.75 μL30U

RNA oligonucleotide

4 μg4μg

TAP-treated or untreated RNA 10 μg10μg

ATP (2 mM) 1.5 μL0.1mM

T4 RNA ligase (20 U/μL) 1.5 μL30U

Oto30μL–

RNA oligonucleotides are at a molar excess of 3–6 over target cellular RNA.

Rapid Ampliﬁcation of cDNA Ends (RACE) 117

ligation. It should look like the previous samples, as dis-

cussed (see Section 3.3, Step 7).

3.5. Reverse

Transcription

1. For each experimental and control sample from Section

3.4, Step 3, assemble the transcription components listed

in Table 8.5 on ice in a sterile microfuge tube.

Table 8.5

Reverse transcription

Component Amount Final

Reverse-transcription buffer (5×)4μL1×

dNTP mixture (containing all four dNTPs,

each at 10 mM)

1 μL 471 μM

DTT (0.1 M) 2 μL 9.41 mM

RNasin (40 U/μL) 0.25 μL10U

Total 7.25 μL–

2. In a separate tube, add 20 ng of a gene-speciﬁc antisense

primer (e.g., GSP-RT to generate the 5



end and the reverse

compliment of NRC3 to generate the 3



end) to the remain-

ing RNA (about 6.7 μg) fr om Section 3.4, Step 3 in 13 μL

of H

O. Incubate for 3 min at 80

◦

C, cool rapidly on ice and

centrifuge for 5 s. To run an optional control reaction, set up

an identical tube in parallel to which no reverse transcriptase

is added in Step 3.

3. Add the RNA-primer mix to the reverse-transcription com-

ponents, subsequently add 1 μL (200 U) of SuperScript II

reverse transcriptase, and incubate for 1 h at 42

◦

Candfor

10 min at 50

◦

4. Inactivate the reverse transcriptase by incubating at 70

◦

Cfor

15 min then centrifuge for 5 s.

5. Destroy the RNA template by adding 0.75 μL(1.5U)

of RNase H to the reaction mixture and incubating it for

20 min at 37

◦

6. Dilute the reaction mixture to 100 μL with TE buffer and

store at 4

◦

C. Note that the 5



end oligonucleotide–cDNA

pool can be stored indeﬁnitely at 4

◦

C. A v oid storing at

–20

◦

C, which could snap some of the cDNA strands with

repeated freeze–thawings.

3.6. First-Round

Ampliﬁcation

1. In a sterile 0.2-mL microfuge tube, mix the reagents listed

in Table 8.6 for each experimental and control sample from

Section 3.5, Step 6.

118 Yeku and Frohman

Table 8.6

First-round ampliﬁcation (reaction pre-mix)

Component Amount Final

Hercules Hot-Start polymerase buffer (10×)5μL1×

dNTP solution (10 mM) 1.0 μL 200 μM

Hercules Hot-Start polymerase 2.5 U 2.5 U

Oto50μL–

2. Add a 1 μL aliquot of the 5



end oligonucleotide–cDNA

pooland25pmoleachofprimersGSP1andNRC1(orthe

reverse compliment of GSP1 and NRC1 if you are cloning

the 3



end). A “no-template” control should also be pre -

pared.

3. Heat in a DNA thermal cycler for 5 min at 98

◦

Ctodenature

the ﬁrst-strand (and to activate the polymerase). Cool for

2 min to the appropriate annealing temperature (56–68

◦

C),

followed by cDNA extension for 40 min at 72

◦

4. Car ry out 35 cycles of ampliﬁcation with a “step” program

as listed in Table 8.7. Products can be stored at 4

◦

Table 8.7

First-round ampliﬁcation (heat-block settings)

Cycle

number Denaturation Annealing

Polymerization-

extension

1–30 10 s at 94

◦

C 10 s at 52–60

◦

C3minat72

◦

31–35 10 s at 94

◦

C 10 s at 52–60

◦

C15minat72

◦

then cool to room

temperature

3.7. Second-Round

Ampliﬁcation

1. Dilute a portion of the ampliﬁcation products from the ﬁrst

round ampliﬁcation 1:20 in TE buffer. Two rounds of ampli-

ﬁcation ensure the speciﬁcity of the yield by quenching the

background ampliﬁcation. The ﬁrst round generates a lot

of background because only one gene-speciﬁc primer is uti-

lized. By using a second gene-speciﬁc primer, the speciﬁcity

of the yield is signiﬁcantly increased.

2. In a sterile 0.2 mL microfuge tube, the reagents listed in

Table 8.8 should be mixed (on ice).

3. Add a 1-mL aliquot of the diluted ﬁrst-round ampliﬁcation

products (obtained in Step 1 above) and 25 pmol each of

Rapid Ampliﬁcation of cDNA Ends (RACE) 119

Table 8.8

Second-round ampliﬁcation (reaction pre-mix)

Component Amount Final

Hercules Hot-Start polymerase buffer (10×)5μL1×

dNTP solution (10 mM) 1.0 μL 200 μM

Hercules Hot-Start polymerase 2.5 U 2.5 U

Oto50μL–

primers GSP2 and NRC2 (or their reverse compliments for

amplifying the 3



end). Set up a “no-template” control as

well.

4. Mix and heat in a DNA thermal cycler for 5 min at 98

◦

Cto

denature the ﬁrst-strand products and to activate the poly-

merase.

5. Car ry out 30 cycles of ampliﬁcation with a “step” program

as described in Table 8.9. The product can be stored at

◦

6. Separate 20% of the products of ﬁrst- and second-round

ampliﬁcation by electrophoresis through a 1% agarose gel.

Speciﬁc partial cDNAs can be checked by Southern blot

analysis. Information gained from this analysis can be used

to optimize the RACE procedure.

Table 8.9

Second-round ampliﬁcation (heat-block settings)

Cycle

number Denaturation Annealing

Polymerization-

extension

1–29 10 s at 94

◦

C 10 s at 52–68

◦

C3minat72

◦

30 10 s at 94

◦

C 10 s at 52–68

◦

C15minat72

◦

then cool to room

temperature

3.8. Anticipated

Results

Depending on the stringency of the ampliﬁcation cycles, the yield

of the desired product could range from less than 1 to 100% of

the ampliﬁed product. After the second set of ampliﬁcations, the

expected yield of speciﬁc products should be about 100%.

If there are alternative transcripts in the starting pool of

mRNA, then several different speciﬁc products should be seen

after the second r ound of ampliﬁcations. These products are iden-

tiﬁed as distinct bands when visualized on an ethidium bromide

stained gel. Upon sequencing, the presence of TATA or CCAAT,

120 Yeku and Frohman

or other promoter elements, and initiator sites around your gene

should identify these products as genuine alternative transcripts.

4. Notes

1. RACE kits are available from Invitrogen, Clontech, and

Ambion. These ready-made systems are user friendly and

powerful. However they are not suited for every purpose.

The investigator might have to optimize the kits using prin-

ciples outlined in this protocol, e.g., using your own gene-

speciﬁc primers (GSP) and/or varying the annealing tem-

perature. The entire procedure can be completed in 3 days

but will take up to 5 days if the pr ocedure is stopped at the

storage points.

2. Adenosine residues are the best “acceptors” for the ligation

of the 3



end of the RNA oligonucleotide to the 5



end of

its target, meaning that the restriction enzyme for lineariza-

tion should be chosen accordingly (for example, NdeI, SalI,

or XhoI, which terminate in an A at the end of the tran-

scription product). A test transcription should be performed

to ensure that all reactions are working properly, and then

the reaction can be further “scaled up.” The oligonucleotide

can be stored indeﬁnitely at –80

◦

C for future experiments. It

is also important to synthesize sufﬁcient oligonucleotide to

allow for losses due to puriﬁcation and “spot-checks” of the

oligonucleotide during use. Alternatively, the DNA or RNA

oligonucleotide can be ordered from a commercial source.

3. 10× T4 RNA ligase buffers supplied by some manufactur-

ers contain too much ATP (25). If the composition contains

more than 1 mM ATP, it is strongly recommended that you

make your own. You can do this by regenerating the com-

ponents used in the supplied buffer and adjusting the ATP

concentration to 1 mM (ﬁnal 1× concentration should be

0.1 mM). Alternatively, the 10× buffer described in r ef (25)

can be used.

4. For the synthesis of the RNA oligonucleotide, it is impor-

tant to select a plasmid that can be linearized at a site

about 100 bp downstr eam from a T7 or T3 RNA poly-

merase site. A plasmid containing an insert cloned into the

ﬁrst polylinker site should be used because primers made

from palindromic polylinker DNA do not amplify well dur-

ing PCR. In the scenario presented here, pBS-SK-GBX-1-



UTR contains the 3



untranslated region of the mouse

Gbx1 gene7 cloned into the SstI restriction site of the

Rapid Ampliﬁcation of cDNA Ends (RACE) 121

plasmid pBS-SK (Stratagene). This plasmid can be linearized

with the restriction enzyme SmaI and transcribed with T3

RNA polymerase to produce a 132-nt RNA oligonucleotide,

although as suggested in Note 2, using a restriction enzyme

that generates a cleavage site that terminates in an A residue

would be preferable. Any other readily ampliﬁable sequence

can be substituted, but avoid ones derived from abundant

endogenous genes (such as actin) to prevent spurious non-

speciﬁc ampliﬁcation.

5. The presence of nonspeciﬁc ampliﬁcation products could

be the result of inappropriate annealing temperatur e. Using

“Touch down PCR” is an effective approach to optimize the

annealing temperature. Alternatively, the same optimization

can be attained by gradually increasing the annealing tem-

perature in 2

◦

C intervals at each r e levant step of the proto-

col.

6. All the primers used for ampliﬁcation are derived from the

RNA oligonucleotide. It is recommended that primer design

is software assisted in order to ensure similar melting tem-

peratures. For instance, Primer3 from the Massachusetts

Institute of Technology (http://frodo.wi.mit.edu/cgi-

bin/primer3/primer3_www.cgi) can be used to generate

suitable candidates.

Acknowledgments

This work was supported by NIH awards GM071520 and

GM084251 to MAF and an NIH T32 Medical Scientist Train-

ing Grant, an F31 National Research Service Award from the

National Institute of Diabetes and Digestive and Kidney Diseases,

and the Turner Foundation award to OY.

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Chapter 9

Fractionation of mRNA Based on the Length

of the Poly(A) Tail

Hedda A. Meijer and Cornelia H. de Moor

Abstract

Poly(A) tail length plays an important role in mRNA stability and translational control. Poly(A)

fractionation is a very powerful technique to separate mRNAs according to the length of the poly(A)

tail. Poly(A) fractionation can be used to detect small changes in poly(A) tail length or to prepare sam-

ples for microarray analysis. RNA or crude lysate is mixed with biotinylated oligo(dT), which is then

bound to paramagnetic streptavidin beads. Oligoadenylated mRNA is eluted ﬁrst with a high salt buffer,

followed by a low salt elution for polyadenylated mRNA. Elution of the RNA in two fractions can be

used as a preparation of samples for microarray analysis while elution of the mRNA in several fractions

can be used to analyse (changes in) poly(A) tail length. This method allows for accurate quantiﬁcation

of the amount of oligoadenylated/polyadenylated RNA in each fraction because it is not dependent on

visualising the smears representing the variations in poly(A) tail length. The method is technically easy,

fast, highly reproducible and can be performed on almost any sample containing RNA.

Key words: Poly(A) tail length, fractionation, translation, mRNA stability, polysome proﬁling.

1. Introduction

The poly(A) tail of an mRNA plays an important role in both

the translational activity and the stability of the mRNA. Most

mRNAs obtain a long poly(A) tail (100–250 nt) in the nucleus,

which gets gradually shortened after exportation to the cytoplasm

(1). However, for some mRNAs the length of the poly(A) tail

is strongly regulated and can therefore be altered dramatically.

These mRNAs can be polyadenylated and/or deadenylated in the

cytoplasm. mRNA deadenylation is thought to precede mRNA

degradation in most cases (2). However, an mRNA with a short

H. Nielsen (ed.), RNA, Methods in Molecular Biology 703,

123