Nielsen H. RNA: Methods and Protocols

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124 Meijer and de Moor

poly(A) tail (oligoadenylated RNA) is not necessarily unstable (3).

The length of the poly(A) tail is also thought to be linked to the

translational activity of that mRNA. A long poly(A) tail seems to

coincide with active translation while a short poly(A) tail corre-

lates with translational repression (4).

One of the most challenging parts of studying poly(A) tail

length is that all methods that are dependent on visualisation

of the size of the poly(A) tail by gel electrophoresis tend to

underestimate the length of the poly(A) tail because the signal

is spread out over a large area which weakens the signal. Most

traditional techniques are based on either northern blotting (for

long mRNAs in combination with RNase H cleavage) or PCR.

The northern blotting approach can only be used for r e latively

abundant mRNAs (5). Several PCR-based protocols (6–8)have

improved detection limits but still suffer from an underestimation

of long poly(A) tails. Both northern blotting and the PCR-based

protocols require the preparation of a sample treated with RNase

H in the presence of oligo(dT) to remove the poly(A) tail as a

marker (5). For some systems, such as Xenopus oocytes, it is pos-

sible to inject radiolabelled reporter mRNAs which can then be

analysed by gel electrophoresis (9). The use of radiation and the

possibility to use very short probes limits the underestimation of

the poly(A) tail length but unfortunately cannot be used in many

experimental systems.

When studying poly(A) tail changes on a global scale, mRNAs

need to be separated on the basis of the length of their poly(A)

tail. The resulting fractions can then be used for microarray anal-

ysis. mRNA can be bound to poly(U) agarose and then eluted at

different temperatures (10). While this technique can be used to

separate the RNA into fractions dependent on poly(A) tail length,

it is labour intensive and difﬁcult to get this technique working

reproducibly.

The protocol detailed here can be used for both the analy-

sis of poly(A) tail length, especially for changes in poly(A) tail

length, as well as for the fractionation of mRNA based on the

length of the p oly(A) tail as a preparation of samples for microar-

ray analysis (see Fig. 9.1). Total RNA or crude lysate is mixed

with a radiolabelled and polyadenylated probe (see Fig. 9.2), and

biotinylated oligo(dT). The probe allows for visualisation of the

fractionation and is furthermore used to estimate the poly(A) tail

length in the sample R NA. The poly(A+) mRNA will bind to the

biotinylated oligo(dT), which in turn is bound to paramagnetic

streptavidin beads. The amount of biotinylated oligo(dT) needs

to be optimised for each sample type (see Fig. 9.3). A magnet is

used to separate the bound mRNA from the unbound RNA with-

out a poly(A) tail or a poly(A) shorter than 15 nt. The poly(A+)

mRNA is then eluted from the beads by decreasing the salt con-

centration, which results in elution of mRNAs with increasing

Fractionation of mRNA Based on the Length of the Poly(A) Tail 125

Unbound RNA

AAAAA

TTTTT

AAAAA

Oligoadenylated RNA

AAAAAAAAAA

Polyadenylated RNA

AAAAAAAAAA

5’UTR ORF 3’UTR

AAAAA

AAAAAAAAAA

TTTTT

AAAAAAAAAA

TTTTT

AAAAAAAAAA

TTTTT

AAAAAAAAAA

TTTTT

Fig. 9.1. Schematic representation of the poly(A) fractionation protocol. Total RNA is

mixed with biotinylated oligo(dT) in a GTC buffer (Step 1). The biotin is then allowed to

bind to the streptavidin paramagnetic beads (Step 2). The unbound fraction is collected

(Step 3) and the beads are washed in 0.5× SSC. The oligoadenylated (Step 4)and

polyadenylated RNA (Step 5) are then eluted in two or more steps by decreasing the

SSC concentration in the elution buffer. A radiolabelled and polyadenylated probe is

added to the RNA sample prior to fractionation. This probe is co-fractionated and allows

for analysis of the fractionation and estimation of the poly(A) tail length of the RNA in the

different fractions. UTR = untranslated region, ORF = open reading frame, B = biotin,

S = paramagnetic streptavidin bead.

poly(A) tail length. For the analysis of (changes in) poly(A) tail

length the mRNA is eluted in 6 fractions (see Fig. 9.4). For the

preparation of samples for microarray analysis the mRNA is eluted

in 2 fractions, (oligoadenylated and polyadenylated mRNA) (see

126 Meijer and de Moor

marker

5.0x

7.5x

10x

15x

20x

25x

30x

40x

no E - pap

E - pap dilution

probe

A24

A42

A566

A70

A168

A255

Fig. 9.2. Polyadenylation of radiolabelled probe. A radiolabelled probe was incubated

with several dilutions of E-PAP. 0.5 μL of marker, 0.5 μL of probe and 1 μL of each

polyadenylation reaction was analysed on a 5% acrylamide/urea/TBE gel. The length of

the marker bands is indicated as the number of nucleotides in excess over the unadeny-

lated probe (indicated with A0, actual length 79 nt).

A24

A42

A566

A70

A168

A255

marker

total

unbound

0.075x SSC

water

800 400 200 100

pmole oligo dT

Fig. 9.3. Poly(A) fractionation into two fractions with different amounts of biotinylated

oligo(dT). 80 μg of total RNA from NIH3T3 cells was mixed with polyadenylated radio-

labelled probe and different amounts of biotinylated oligo (dT) and separated using the

poly(A) fractionation protocol. RNA was eluted with 0.075× SSC (oligoadylated RNA)

and water (polyadenylated RNA). This shows that 800 pmole of oligo dT is required for

efﬁcient fractionation of 80 μg of RNA from NIH3T3 cells.

Fig. 9.3). Since poly(A) tail length and translational activity are

thought to be correlated for many mRNAs, poly(A) fractionation

can be used as an alternative for other posttranscriptional proﬁling

methods. Poly(A) fractionation is a fast and reproducible method

which can be used for a wide variety of samples such as tissue cul-

ture cells, oocytes, embryos, clinical samples and total RNA (3).

Fractionation of mRNA Based on the Length of the Poly(A) Tail 127

marker

A24

A42

A566

A70

A168

A255

total

unbound

0.2x SSC

0.1x SSC

0.075x SSC

0.050x SSC

0.025x SSC

water

elutions

Fig. 9.4. Poly(A) fractionation into 6 f ractions. 80 μg of total RNA from NIH3T3 cells was

mixed with polyadenylated radiolabelled probe and 750 pmole of biotinylated oligo(dT)

and separated using the poly(A) fractionation protocol. The marker can be used to esti-

mate the range of poly(A) tail length in each fraction. Note that the signal in the water

lane is weak due to the lack of probe with a very long poly(A) tail.

2. Materials

2.1. Molecular

Weight Marker

1. Linearised plasmid DNA. The marker is a mixture of radioac-

tively labelled in vitro transcripts of known lengths. In order

to make this, you will need several plasmids which dif-

fer slightly in size when digested with the same restriction

enzyme. Alternatively, you can use one plasmid which can

be digested by several different restriction enzymes (one at a

time).

2. The same materials as for the puriﬁcation of the template (see

Section 2.2) and for the probe synthesis (see Section 2.3).

2.2. Template

Puriﬁcation

1. Linearised plasmid DNA (this plasmid will also generate the

lowest band of the molecular weight marker).

2. Water-saturated phenol.

3. Chloroform.

4. 3 M NaAc, pH 5.2.

5. 100% isopropanol.

6. 70% ethanol.

7. RNase-free water.

128 Meijer and de Moor

2.3. Probe Synthesis

1. RNA polymerase (including 5× transcription buffer (TSC)

and 100 mM dithiothreitol (DTT)) (Promega).

2. NTP mix: 5 mM ATP, 5 mM CTP, 0.1 mM UTP,

0.5 mM GTP.

3. 40 U/μL RNasin.

4. 5 mM cap analogue (m

GpppG) (Ambion).

5. 1 μg/μL template in R Nase-free water.

6. 800 Ci/mmole, 20 μCi/μL[α-

P]UTP.

7. Water-saturated phenol.

8. Chloroform.

9. G-50 Sephadex in water, autoclaved: store at 4

◦

10. 1-mL syringes.

11. Sterilised and silanised glass wool.

2.4. Polyadenylation

1. Poly(A) Tailing Kit (containing 2 U/μL Escherichia coli

poly(A) polymerase (E-PAP), 5× E-PAP buffer, 10 mM

ATP , 25 mM MnCl

, nuclease free water) (Ambion).

2. RNase-free TE: 10 mM Tris-HCl, 1 mM ethylenediamine

tetraacetic acid (EDTA) (pH 8.0), autoclaved.

3. Water-saturated phenol.

4. Chloroform.

2.5. Analysis

of Probes

1. Urea.

2. RNase-free 10× TBE: 500 mM Tris-base, 500 mM boric

acid, 1 mM EDTA, pH 8.0; autoclaved.

3. 30% acrylamide/bisacrylamide (37.5:1).

4. 10% ammonium persulfate (APS); fr e shly prepared or

aliquoted and stored at –20

◦

5. N,N,N,N’-tetramethyl-ehylenediamine (TEMED).

6. Gel loading buffer: 95% formamide, 0.025% xylene cyanol,

0.025% bromophenol blue, 18 mM EDTA, 0.025% sodium

dodecyl sulfate (SDS).

2.6. Oligo(dT)-Based

Fractionation of RNA

1. 80 μg of total RNA in RNase-free water.

2. PolyA Tract System 1000, containing paramagnetic

streptavidin beads, guanidinium thiocyanate (GTC)

extraction buffer, dilution buffer, biotinylated oligo(dT),

β-mercaptoethanol (BME), 0.5× SSC and magnetic

separation stand (Promega).

3. Extra 50 p mol/μL biotinylated oligo(dT) (Promega).

4. RNase-free water.

Fractionation of mRNA Based on the Length of the Poly(A) Tail 129

5. 3 M NaAc, pH 5.2.

6. 100% isopropanol.

7. 10 μg/μL yeast tRNA.

8. 70% ethanol.

2.7. Analysis of

Fractionation

1. The same materials as for the analysis of the probes (see

Section 2.5)

3. Methods

(see Note 1)

3.1. Molecular

Weight Marker

Linearise the plasmids as described for the puriﬁcation of the tem-

plate (see Section 3.2) and then mix them and use for the syn-

thesis of the molecular weight marker as described for the probe

synthesis (see Section 3.3)(see Note 2).

3.2. Template

Puriﬁcation by

Phenol Extraction

and Ethanol

Precipitation

1. Linearise the p lasmid DNA.

2. Add 0.5 volume of phenol and vortex for 10 s (see

Note 3).

3. Add 0.5 volume of chloroform and vortex for 30 s.

4. Spin at 20,000 ×g for 10 min at room temperature.

5. Transfer the supernatant to a new tube. Add 1 volume of

chloroform and vortex for 30 s.

6. Spin at 20,000 ×g for 10 min at room temperature.

7. Transfer the supernatant to a new tube. Add 0.1 volume of

3 M NaAc, pH 5.2, and vortex.

8. Add 1 volume of isopropanol and vortex.

9. Incubate on ice for 30 min.

10. Spin at 20,000 ×g for 10 min at 4

◦

11. Aspirate the supernatant. Wash the pellet with 2 volumes

of 70% ethanol.

12. Spin at 20,000×g for 5 min at 4

◦

C and remove residual

ethanol.

13. Dry the pellet and dissolve it in 10 μLofH

14. Check an aliquot of the template on a 1.5% agarose gel.

15. Quantify the template and dilute it to 1 μg/μL.

3.3. Probe Synthesis

by In Vitro

Transcription

1. Mix:1 μL5×TSC

0.5 μL0.1MDTT

0.5 μL NTP mix

0.5 μL40U/μL RNasin

130 Meijer and de Moor

0.5 μL 5 mM cap analogue

0.5 μL template (1 μg/μL)

1.5 μL 800 Ci/mmole, 20 μCi/μL[α-

P]UTP (see

Note 4)

0.25 μL RNA polymerase (see Note 5).

2. Incubate for 1 h at 37

◦

3. In the meantime prepare a G-50 Sephadex column per

sample. Remove the plunger fr om a 1-mL syringe and put

a piece of glass wool in the syringe. Push it down with

the plunger to the bottom of the syringe and then remove

the plunger. Swirl the Sephadex to mix it and pipette it

into the syringe, avoiding air bubbles. Completely ﬁll the

syringe. Place the syringe in a 15-mL Falcon tube. Spin

for 1 min at 1,300×g at 4

◦

C. Discard the ﬂow through.

Keep adding and spinning until the syringe contains 1 mL

of Sephadex. Spin for 5 min at 1,300×g at 4

◦

C. Discard

the ﬂow through and move the syringe to a new 15-mL

Falcon tube. The G-50 Sephadex column is now ready for

the puriﬁcation of the probe.

4. After the incubation add 45 μLofH

O to the probe.

5. Add 25 μL of p henol and vortex for 10 s.

6. Add 25 μL of chloroform and vortex for 30 s .

7. Spin at 20,000 ×g for 10 min at 4

◦

8. Load the supernatant on the G-50 Sephadex column.

9. Spin for 1 min at 1,300×g at 4

◦

10. Transfer the eluate to a 1.5-mL tube.

11. Load 50 μL of RNase-free H

O onto the Sephadex col-

umn.

12. Spin for 1 min at 1,300×g at 4

◦

13. Transfer the eluate to the tube with the ﬁrst eluate. The

combined eluates is the unadenylated probe.

3.4. Polyadenylation

Assemble on ice! (see Note 6)

1. Prepare E-PAP dilutions (7.5×,15×,30× in 1× E-PAP

buffer).

2. For each E-PAP dilution mix:

5 μL5× E-PAP buffer

2.5 μL25mMMnCl2

2.5 μL10mMATP

5 μL diluted E-PAP

10 μL unadenylated probe (see Section 3.3)

3. Incubate for exactly 30 min at 37

◦

4. Immediately put samples on ice.

Fractionation of mRNA Based on the Length of the Poly(A) Tail 131

5. Add 25 μL of TE and mix.

6. Add 25 μL of p henol and vortex for 10 s.

7. Add 25 μL of chloroform and vortex for 30 s.

8. Spin at 20,000 ×g for 10 min at 4

◦

9. Transfer the supernatant to a new tube. The polyadenylated

probe is used without further puriﬁcation steps or concen-

tration by ethanol precipitation.

3.5. Analysis

of Probes on a 5%

Acrylamide/Urea/TBE

Mini Gel

1. Mix:

6gurea

1.2 mL 10×TBE

2 mL 30% acrylamide/bisacrylamide (37.5:1) (see Note 7)

4mLH

2. Microwave brieﬂy to dissolve urea. Do not allow the solu-

tion to get warmer than 60

◦

3. Let the solution cool down to room temperature.

4. Add 24 μL of APS and 24 μL of TEMED (see Note 8).

5. Pour the gel and allow it to set.

6. Pre-run the gel at 100 V for 15 min.

7. Prepare samples:

0.5 μL marker in gel loading buffer

0.5 μL pr obe w/o poly(A) tail in gel loading buffer

1 μL polyadenylated probe (30× diluted E-PAP) in gel

loading buffer

1 μL polyadenylated probe (15× diluted E-PAP) in gel

loading buffer

1 μL polyadenylated probe (7.5× diluted E-PAP) in gel

loading buffer.

8. Denature the samples at 95

◦

Cfor5min.

9. Load the samples and run at 100 V (until bromophenol

blue is almost at the bottom of the gel).

10. Wrap the gel in Saran wrap and expose it to a PhosphoIm-

ager screen or ﬁlm for 5–30 min.

11. Mix the polyadenylated samples to produce a range from

A0 to A400, including a small amount of probe without

poly(A) tail. This constitutes the probe mix (see Fig. 9.2).

3.6. Oligo(dT)-Based

Fractionation of RNA

All steps ar e at room temperature unless indicated otherwise.

1. Allow the GTC, BME, biotinylated oligo(dT), 0.5×SSC

and H

O to r each room temperature.

2. Add 41 μL of BME per mL of GTC (GTC/BME).

3. Add 20.5 μL of BME per mL of dilution buffer

(DIL/BME). Preheat the DIL/BME to 70

◦

132 Meijer and de Moor

4. Make the required SSC dilutions (0.200×, 0.100×,

0.075×, 0.050×, 0.025×).

5. Mix a maximum of 40 μLtotalRNA(max80μg) with

400 μL GTC/BME in a 2-mL tube (see Note 9).

6. Add 5 μL of polyadenylated probe mix (see Section 3.5),

15 μL biotinylated oligo(dT) and 816 μL DIL/BME

(see Note 10,andFig. 9.3).

7. Incubate at 70

◦

Cfor5min.

8. Spin at 12,000 ×g for 10 min at room temperature.

9. In the meantime prepare the paramagnetic streptavidin

beads. Completely resuspend the beads by gently rock-

ing the bottle. Transfer 600 μL of resuspended beads to a

2-mL tube for each sample. Place the tube on the magnetic

stand and slowly move the stand to a horizontal position

until the beads are collected at the tube side. Carefully pour

off the storage buffer by tilting the tube so that the solu-

tion runs over the captured beads. Resuspend the beads in

600 μL0.5×SSC and capture the beads again using the

stand. Repeat this wash step twice. Resuspend the beads in

600 μL0.5×SSC.

10. After the spin add the supernatant to the beads. Allow the

biotinylated oligo(dT) to bind to the beads by rotation at

room temperature for 15 min.

11. After the biotinylated oligo(dT) has bound to the beads

capture the beads using the magnetic stand. Transfer the

supernatant to a new tube and keep on ice (unbound frac-

tion).

12. Wash the beads three times as described above (See

Step 9). Rotate for at least 5 min between each wash.

13. Resuspend the beads in 400 μL 0.200× SSC. Rotate for at

least 5 min at room temperature. Capture the beads using

the magnetic stand. Transfer the eluate (400 μL) to a new

tube. Be careful not to disturb the beads when doing so.

Keep the 400 μL in a tube on ice (eluate 1) (see Note 11).

14. Repeat the elution (Step 13) with 400 μL 0.100× SSC,

0.075× SSC, 0.050× SSC, 0.025× SSC and H

O (eluates

2–6) (see Note 11).

15. Spin all collected fractions (unbound and eluates 1–6) at

20,000×g for 5 min at 4

◦

C to remove any transferred

beads.

16. Transfer 2× 650 μL of the unbound supernatant to two

new tubes. Add 650 μL of isopropanol to each. Mix and

precipitate overnight at –20

◦

Fractionation of mRNA Based on the Length of the Poly(A) Tail 133

17. Transfer 360 μL of each eluate supernatant to a new tube.

Add 2 μL yeast tRNA, 36 μL 3 M NaAc pH 5.2 and

360 μL isopropanol. Mix and precipitate overnight at –

◦

18. Spin all samples at 20,000×g for 30 min at 4

◦

19. Remove the supernatant and wash with 800 μL 70%

ethanol.

20. Spin at 20,000 ×g for 10 min at 4

◦

21. Completely remove the supernatant with a drawn out glass

pipette and allow the pellets to dry.

22. Resuspend the pellets from the eluates in 10 μLofH

Resuspend the pellets from the unbound fraction in 5 μL

of H

O per tube and pool the two samples.

3.7. Analysis of the

Fractionation on a

5% Acrylamide/

UREA/TBE Maxi Gel

1. Mix per gel:

30 g urea

6mL10×TBE

10 mL 30% acrylamide/bisacrylamide (37.5:1)

20 mL H

2. Microwave brieﬂy to dissolve urea. Do not allow the solu-

tion to get warmer than 60

◦

3. Let the solution cool down to room temperature.

4. Add 120 μL of APS and 120 μL of TEMED.

5. Pour the gel and allow to set.

6. Pre-run the gel at 200 V for 15 min.

7. Prepare samples:

1 μL of molecular weight marker 20× diluted in gel load-

ing buffer.

0.9 μL of probe mix in gel loading buffer.

1 μL of unbound 5× diluted in gel loading buffer.

1 μL for each eluate in gel loading buffer (see Note 12).

8. Denature the samples at 95

◦

Cfor5min.

9. Load the samples and run at 200 V (until the dark blue

colour of the dye indicator is almost at the bottom of the

gel).

10. Wrap the gel in Saran wrap and expose it to a PhosphoIm-

ager screen or ﬁlm for 24 h at –20

◦

C. Allow the cassette to

reach room temperature before opening (see Fig. 9.4).

3.8. Further Analysis

The remainder of the samples can be used for subsequent analysis

by RT-qPCR and/or northern blotting to show in which fractions

endogenous and/or reporter RNAs are (see Note 13).