Subtrmethods

Subtractive Probe Analysis

For comparisons between embryonic stages, tissues, or cell types; or for use with differential, subtractively prepared complex probes in which transcripts present under one condition and not others are preferentially represented, it is essential to be able to probe the greatest variety of relevant cDNAs. Therefore in sea urchin embryos, for which there are yet only a few thousand EST sequences, large macroarray libraries are the obvious choice. However, to use macroarrays successfully for differential screening it is necessary to deal with the relatively insensitive, high volume hybridization conditions required for macroarray filter screens. As described below, we have already make excellent progress on developing technologies to accomplish this objective.


To improve sensitivity we utilize single-stranded RNA probes transcribed by T7 polymerase from primer sites incorporated in the cDNA primers, and instead of a few ng we use 1-2µg of probe per 30-50ml (for hybridization with several filters at once). Under these conditions about 25% of spots react. For sea urchin embryos this means that without further increases in sequence concentration, macroarray screening with complex probes suffices to detect mRNAs present at >30-50 copies per cell. Sequence concentration can be increased markedly for differentially expressed genes by subtractive hybridization. We have developed an elegant procedure for this purpose in which single-stranded driver RNA is used to trap out sequences held in common between the two transcript populations (Rast, J. P., Amore, G., Calestani, C., Livi, C. B., Ransick, A. and Davidson, E. H. Recovery of developmentally defined gene sets from high-density cDNA macroarrays. Dev. Biol. 228, 270-286, 2000). A detailed version of the protocol described in the paper is presented below. The end result is that we can now detect sequences down to ten or so copies per average cell.

I. COMPLEX PROBE SYNTHESIS

A. TOTAL RNA ISOLATION

1. 300-5000 sea urchin embryos or an equivalent mass of embryo cells are centrifuged from filtered sea water at 5000 x g and the resulting pellet is resuspended and lysed in 500 µl of RNAzol (Leedo Medical Laboratories, Houston TX). The samples are then either immediately extracted or the lysate is frozen on dry ice and stored at -70°C to be processed later.

2. A one-tenth volume (50 µl) of chloroform is added. The sample is then vortexed for 15 seconds and placed on ice for 5 minutes followed by centrifugation in a microfuge at 14,000 x g for 10 minutes.

3. The upper aqueous phase (~250 µl) is transferred to a clean tube. If fewer than 1000 embryos or
equivalent numbers of dissociated cells are being extracted then 5µg of RNase-free glycogen is added to the sample as carrier. One volume of isopropanol is added and the sample is placed at − 20°C for 2 hours to overnight.

4. The sample is then centrifuged for 20 min. at 4°C at 14,000 x g. The RNA pellet is washed with 200 µl 75% ethanol, recentrifuged and after removal of wash the pellet is air dried for ~10 minutes. The RNA pellet is then resuspended in 25 µ l RNase-free dH₂O.

B. POLYADENYLATED RNA ISOLATION

Polyadenylated RNA was isolated on Poly-T₂₅ magnetic beads (Dynal Inc., Lake Success, NY) as per the instructions of manufacturer except that the isolation was scaled down to use 50 µ l (250 µg ) of beads.

1. 50 µl of beads are washed with 50 µl of 2X BINDING BUFFER (1 M LiCl; 20 mM Tris, pH 7.5; 2 mM EDTA),magnetically collected and resuspended in 25 µl 2X BINDING BUFFER.

2. 25 µl of RNA is heated to 65°C for 2 minutes then mixed with the beads at room temperature.
The beads are kept resuspended for 10 minutes by gentle inversion to allow mRNA annealing (the liquid should remain at the conical end of the Eppendorf tube during this procedure).

3. The beads are magnetically collected, the 2x BINDING BUFFER is removed and 50 µl WASH BUFFER (0.15 M LiCl; 10 mM Tris pH 7.5; 1 mM EDTA) is added. The wash procedure is then repeated.

4. After carefully removing all of the WASH BUFFER the beads are resuspended in 10 µl of RNase-free dH₂O, the sample is heated to 65°C for 2 minutes, the beads are magnetically collected and the eluted mRNA is removed to a clean tube.

5. The volume of the RNA sample is then reduced to 2.5 µl in a Speed-Vac.

C. cDNA SYNTHESIS

cDNA is sythesized using enzymes and bufferes taken from a Clontech Marathon cDNA sythesis kit. Half-reactions are used when less than 500 ng of mRNA is used as template. Qiagen PCR purification columns are used to purify samples between synthesis stages.

1^st-Strand DNA synthesis

1. 0.5 µl LT7RND-BT Primer (0.5µg/µl) is added to the mRNA (\243500 ng) in an 0.2 ml PCR tube and the mixture heated to 65°C for 2 min. in a PCR machine then chilled on ice.LT7RND-BT: 5'-(biotin)-CGGAGGTAATACGACTCACTATAGGGAGNNNNNN-3' (34 nt).
2. 1µl of 5X CLONETECH FIRST STRAND BUFFER (250 mM Tris, pH 8.5; 40 mM MgCl₂; 150 mM KCl; 5mM DTT), 0.5 µl of a 10mM each dNTP solution, and 0.5 µl AMV reverse transcriptase (20U/µl) is added.
3. The sample is incubated at room temperature for 10 min., then at 42°C in an air incubator for 1 hour.
4. 200 µl of Qiagen buffer PB is added and the sample is processed through a Qiagen PCR cleanup column according to the manufacture’s instructions to remove remaining LT7RND-BT oligonucleotide. The
   

sample is eluted in 30 µl of 1/2 x EB (5 mM Tris, pH 8.5).

2^nd-Strand DNA synthesis and blunt ending

1. The sample is placed on ice and 8 µl of clontech 5X 2^nd STRAND BUFFER (500mM KCl; 50mM
   

(NH₄)₂SO₄; 25 mM MgCl₂; 0.75 mM b -NAD; 100mM Tris, pH 7.5; 0.25 mg/ml BSA), 1.2 µl 10 mM dNTP stock and 2 µl 20X 2^nd strand enzyme cocktail (E. coli DNA pol 1, 6U/µl; E coli DNA ligase, 1.2 U/µ l; E coli RNase H, 0.25U/µl) is added. The reaction is incubated 1.5 hr. at 16°C.

1. 1 µl T4 DNA pol (5U/µl) is added and the sample is incubated a further 45 min. at 16°
   

C. 200 µl of Qiagen PB solution is added and the sample is placed in a Qiagen PCR cleanup column, processed according to protocol and eluted in 50 µl EB.

1. Next 2.2 µl of 20X EDTA/Glycogen (200mM EDTA, 2 µg/µl glycogen), 26 µl 4M NH₄OAc and 192.5 µl room temperature ethanol is added and the sample is mixed and centrifuged at 14,000 rpm for 20 min. at room temperature. The pellet is washed by the addition of 150 µl 80% ethanol centrifuged again for 10 min, the supernatant is removed and the sample is air dried for ~10 min.

D. Linker Ligation

Different linkers are used for cDNA to be used as Selectate or Driver to avoid non-specific subtraction.

For Selectate:

1. The sample is resuspended in 5 µl dH₂O. This is combined with 2 µl of a 150 ng/µl Stock of annealed linker, 2 µl 5X ligase buffer and 1 µl ligase. The sample is then incubated at 16°C for ~16 hrs.

S-LINKER

5’- GGGTGCTGTATTGTGTACTTGAACGGGCGGCCGCA-3’ (35nt)

3’- DIDEOXY-(C)GCCCGCCGGCGT-P- 5’(12nt)

2. After ligation the sample is diluted in 200 µl PB buffer and processed though a Qiagen PCR cleanup column. The sample is eluted in 50 µ l EB.

Driver cDNA is synthesized just as selectate cDNA except that(1) no size selection is performed, (2) only 3-5 cycles of PCR are used and (3) a different linker (below) is used to avoid linker driven hybridization to selectate.

D-Linker

5' - GCCAACGTATGTAAGGTTGAGTTCCGGGCAGGT-3'

3'- ddCCCGTCCA- P-5'

E. cDNA AMPLIFICATION

1. 2.5 µl of sample is employed as template in a 25 µl PCR using the 125 ng of each of the LT7PRIMER and LINKPRIMER oligonucleotides:

The sample is cycled as follows:

Then to: 4°C

From this pilot PCR the lowest cycle number sufficient to generate 100-200 ng of cDNA starting with 25µl (1/2) of the 1° cDNA is estimated and a second PCR is performed in a 100 µl volume.

This reaction is cleaned up in a Qiagen PCR column and the entire sample is electroporesed on a 1.5 % agarose gel. The region corresponding to 500-700 bp is excised and the cDNA isolated using a Qiagen Gel extraction column and eluted in 50µl EB.

A second PCR titration is set up as in step (1) except that the LT7PRIMER is replaced by the BT-LT7PRIMER. The PCR is optimised so that a 25 µl reaction will produce 500-700 ng of product assessed spectrophotographically after Qiagen PCR column removal of small products and remaining primers.

The optimised parameters are used to produce ~3µg product with minimal cycle number.

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II. PREPARATION OF SINGLE−STRANDED SELECTATE AND DRIVER

1. SELECTATE: ~3 µg of size selected cDNA is bound to Dynal Streptavidin beads and the (+) strand is eluted by alkaline treatment, precipitated and quantified spectrophotometrically. This should produce ~1 µg single stranded cDNA.

1. 3µg biotinylated cDNA is diluted into 100 µl 6’SSC.
2. 100 µl Dynal Streptavidin beads (M-280) (=1mg beads; 6.5 µg binding capacity) are washed once with 400 µl 6’SSC, magnetically collected and resuspended in 100 µl 6’SSC.
3. The beads are again magnetically collected, then resuspended with the 100 µl Bt-cDNA solution from step (a). The bead suspension is incubated at 43°C with gentle agitation for 45 minutes after which the beads are recollected on a magnet and the supernatant is removed and saved for analysis.
4. The beads are then washed 2’ with 400µl 1’SSC at room temperature and once with 400 µl TE (10 mM Tris pH 7.5, 1 mM EDTA) to remove unbound cDNA.
5. The beads are then resuspended in 50 µl Melting Buffer (0.125 M NaOH, 0.1 M NaCl) and incubated at room temperature for 10 minutes. The beads are magnetically recollected and the supernatant with eluted (+) strand is retained.
6. 11 µl of 10’TE (100mM Tris, 10 mM EDTA, pH 7.5) and 12 µl of neutralisation buffer (1.25 M acetic acid) is added to the supernatant from (e).
7. The single stranded selectate DNA is precipitated with the addition of 8.2 µl 3 M NaOAc and 262 µl EtOH. After 2hrs at -20°C the sample is microfuged for 20 min. Following removal of the
   supernatant, 100 µl of cold 75% EtOH is added the sample is again microfuged for 5min. The ethanol wash is then removed and the sample is dried at room temperature and resuspended in 20 µl DEPC dH₂O.
8. 3µl of the selectate is diluted in 60µl dH₂O and the sample is quantified on a spectrophotometer

2. DRIVER: For streptavidin capture biotinylated RNA is used as driver (2a). For Hydroxylapatite (HAP) separation nonbiotinylated (-)-strand RNA is used (2b). HAP separation is the more efficient of the two methods.

2a. DRIVER FOR STREPTAVIDIN CAPTURE

1. The sample is incubated 6 hrs. to overnight at 37°C.
2. 1 µl DNase (RNase-free; 2U/µl) is added and the sample is incubated a further 1 hr.
3. The sample is then incubated at 80°C for 5 min.
4. 30 µl DEPC dH₂O and 25 µl 7.5 M LiCl, 50 mM EDTA is added and the sample is placed at - 20°C for 2 hrs.
5. The RNA is centrifuged for 20 min. at 4°C, washed with 75% EtOH, air dried and dissolved in 30 µl DEPC dH₂O. Care must be taken to ensure complete dissolution.
6. 1 µl in 60 µl dH₂O is quantified spectrophotometrically. DEPC dH₂O is added to achieve a final concentration of 2.5µg/µl.

2b. DRIVER FOR HAP SEPARATION

1. a. The sample is incubated 6 hrs. to overnight at 37° C.
2. 1 µl DNase (RNase-free; 2U/µl) is added and the sample is incubated a further 1 hr.
3. The sample is then incubated at 80°C for 5 min.
4. 30 µl DEPC dH₂O and 25 µl 7.5 M LiCl, 50 mM EDTA is added and the sample is placed at -20°C for 2 hrs.
5. The RNA is centrifuged for 20 min. at 4°C, washed with 75% EtOH, air dried and dissolved in 30 µl DEPC dH₂O. Care must be taken to ensure complete dissolution.
6. 1 µl in 60 µl dH₂O is quantified spectrophotometrically. DEPC dH₂O is added to achieve a final concentration of 2.5µg/µl.

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III. SUBTRACTIVE HYBRIDIZATION

Two methods for subtractive hybridization are outlined. The first is modified from (Sagerstrom, C. G., Sun, B. I., and Sive, H. L., 1997) and uses streptavidin capture/phenol extraction to separate unhybridized selectate. The second method uses hydroxylapatite column chromatography (for review see Britten et al., 1974). The HAP method requires more equipment and setup effort, but is more efficient at removing hybrids with minimal loss of unique single stranded sequence.

A. STREPTAVIDIN CAPTURE/PHENOL EXTRACTION

Hybridizations are set up as follows. If materials permit 20µl hybridizations can be set up with 10 −20 µg Driver RNA and up to 1/10 mass of selectate (100-200 ng).

1. Selectate and driver are mixed in 10 µl dH₂O and the sample is denatured at 95°C for 5 minutes then placed on ice.

2. 10 µl of 65°C 2’ Hybridization Buffer is then added and the sample is placed in a
pre-warmed 65°C humidity chamber (a 50 ml Falcon tube with a small piece of moistened tissue paper works well). This is then placed in a 65°C air incubator for 24 hours.

2’Hybridization Buffer (10 ml)

50 mM HEPES pH 8.0 500 µl 1M HEPES

10 mM EDTA 200 µl 0.5M EDTA

0.2% SDS 100 µl 20% SDS

1.2 M NaCl 2.4 ml 5M NaCl

0.2% PPi 400 µl 5% PPi

6.4 ml dH₂O

Removal of Selectate-Driver Hybrids

3. 150 µl of Extraction Buffer is added on ice then 15 µl (2ug/µl) streptavidin is added, the sample is incubated on ice for 1 hr. The sample is then placed at 68°C for 10 min. after which it is again placed on ice.

10ml Extraction Buffer (for 20µl hybridization)

1 ml 1M HEPES pH 8.0

20 µl 0.5M EDTA

21.4 µl 5M NaCl

20 µl 5% PPi

8.94 ml dH₂O

4. Extraction is performed in a cold room and on ice to minimise the effects of phenol:water emulsion on hybridisation.165 µl of ice cold phenol chloroform (1:1; pH=8.0) is added and the sample is vortexed for exactly 5 seconds, then immediately placed in a centrifuge and spun at full speed for five minutes. The
aqueous layer is removed and the sample is re-extracted with chloroform.

B. HYDROXYLAPATITE COLUMN CHROMATOGRAPHY

Hydroxylapatite (HAP) column chromatography was used to improve isolation of non-hybridized single-stranded selectate (for general review of HAP chromatography methods, see Britten et al., 1974).

For these hybridizations reactions are set up as follows:

1. 10 ¼g of (-)-strand RNA driver and 200 ng of (+)-strand is placed in 10¼l dH₂O, denatured at 95\272C for 5 minutes and placed on Ice.

2. The sample is warmed to 65\272C and 10 ¼l of 65\272C 2X HAP Hybridization Buffer (2X=0.68 M PB, 0.02% SDS) is added to the sample.

3. The sample is hybridized in an air incubator at 65\272C for 40 hours.

After hybridization of driver and selectate, HAP chromatography was performed at a temperature and salt criterion at which double-stranded nucleic acid binds to the column while single- stranded is eluted.

1. HAP is equilibrated in 0.12M PB, 0.05% SDS and a 200 ul bed column is prepared on a water-jacketed column maintained at 60 C. All solutions are warmed at 60 C.

2. Column is washed with 10 volumes of 0.12M PB, 0.05% SDS before loading the sample.

3. Sample is adjusted at a salt concentration of 0.12 M PB and a volume of 200ul and maintained at 60 \272C.

4. Sample is loaded and eluate is collected.

5. Column is washed with 9 volumes of PB 0.12 M, 0.05% SDS.

The 2 ml eluate is desalted and concentrated with a Centricon YM-10 spin filter (Millipore, Bedford, MA).

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IV. LIBRARY SCREENING WITH COMPLEX PROBES

A. Probe Labeling

Probe synthesis is performed with an Ambion Maxiscript kit and Amersham 800Ci/mM ³²P-UTP. Probe reaction is set up at room temperature as follows:

Template cDNA 300* ng in 16.8 µl dH₂O

4.8 µl 10’ Transcription buffer

2.4 µl 2 mM UTP (cold)

2.4 µl 10 mM rCTP

2.4 µl 10 mM rATP

2.4 µl 10 mM rGTP

12 µl 32P-UTP 800Ci/mM,20µCi/µl\206

4.8 µl T7 RNA Pol

The reaction is incubated at 37°C for 2hrs, then 2 µl RNase-free DNase is added and the reaction is incubated a further 30 min at 37°C. The reaction is then passes over an RNase-free G50 column and 1µl of a 1/20 is counted to estimate incorporation.

100% incorporation = 5.36 µg @ 1.37’10⁸ cpm/µg. The reaction typically yields 2-2.5 µg of probe.

• Probably as little as 50 ng in adequate.

\206Use Amersham ³²P-UTP as early as possible before calibration date.

B. Filter Hybridization

Four to six filters are prehybridized in ~100 ml 5XSSPE; 5% SDS; 0.1% NaPPi (same as hybridization solution) for ~2hrs hours at 65°C.

1X SSPE = 0.15 M NaCl, 10 mM phosphate, 1 mM EDTA, pH 7.4

Approximately 60 ml of hybridization solution is necessary to wet 5 filters with spacers.

After pouring off prehybridization solution, 15 to 20 ml of fresh solution is added to the wetting volume. The RNA probe is heated to 95°C for 5 min prior to adding to filter. The filters are hybridized with probe for 48 hours at 65°C.

Filters are washed:

(1) 2X 20 min at room temperature in 2xSSPE; 0.1% SDS; 0.05% NaPPi

(2) 2X 20 min at 65°C in 1 x SSPE; 0.1% SDS; 0.05% NaPPi

(3) 1X 15 min at 65°C in 0.1 x SSPE; 0.1% SDS; 0.05% NaPPi

They are then immediately sandwiched between two layers of Saran Wrap and exposed to a Phosphoimager screen for ~24 hrs. Record exact time of exposure. Calibrate longer exposure so that the darkest spots nearly saturate the phosphor screen.

REFERENCES

Britten, R.J., Graham, D.E., Neufeld, B.R.(1974). Analysis of repeating DNA sequences by reassociation.Meth. Enzymol. 29, 363-418.

Sagerstrom, C. G., Sun, B. I., and Sive, H. L.(1997). Subtractive cloning: past, present, and future. Ann. Rev. Biochem. 66, 751-783.

Revised: 11/20/00

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