Synthesis of double-stranded cDNA

T otal RNA was extracted w ith TRIZOL (GibcoBRL) and D N ase-treated, as described above (2.3.1). RDA requires the synthesis of doubled-stranded cDNA (ds- cDNA), in contrast w ith the single-stranded cDNA used for RT-PCR (2.3.2). SMART cDNA Synthesis kit (Clontech) provides aU the reagents necessary for double­ stranded cDNA production.

The generation of first-strand cDNA uses the C ap-finder method, described above for virtual N orthern blots (2.3.4.3). Previous m ethods of generating ds-cDNA relied on RNaseH, a DNA polymerase and a DNA ligase, resulting in relatively poor yields. However, w ith the CapFinder technique, the second cDNA strand can be synthesised by PCR. In addition, the cDNA can be amplified further by a low cycle num ber PCR reaction resulting in workable am ounts of cDNA from limiting starting material.

To generate ds-cDNA for RDA, the cycle num ber for the PCR reaction (below) m ust be titrated. A cycle num ber betw een 16 and 22 is usually required, w ith the correct cycle num ber being the one that gives a clear cDNA sm ear on an ethidium brom ide staining agarose gel from 6 kb- 2 0 0 bp, while still displaying a linear

amplification of material w hen compared to the products obtained from a PCR using the previous cycle num ber. For m ost tissues, distinct bands should be visible in the smear, representing abundant mRNA transcripts.

PCR samples contained: 5 pi PCR buffer (lOx), 41 pi dH^O, 1 pi dNTPs (10 mM), 1 pi EXT-primer (100 n g /p i), 1 pi ss-cDNA and 1 pi A dvantage 2 DNA polym erase (Clontech)

EXT-primer, 5-AAGCAGTGGTAACAACGCAGAGT

Reactions were ru n on a PTC-225 DNA engine (MJ Research), for 2 m in 95°C and then 16-21 cycles of: 15 sec at 95°C, 30 sec at 65°C and 6 m in 6 8°C.

To create a tester representation, 4 reactions w ere done; to generate a driver representation, 24 reactions were set up.

Each 4 PCR reactions were pooled (total volum e = 200 pi) and subjected to one phenol / chloroform extraction, followed by one chloroform extraction. For cDNA

precipitation, 20 pi of NaOAc 2 M and 220 pi of isopropanol w ere added to the final aquous phase and this was incubated at RT for 20 min. (Note: it is critical that isopropanol and not ethanol is used at this stage.) After centrifugation at 16,000xg for 10 m in, the pellet was washed with 70% ethanol, dried, and resuspended in 34 pi

dUp.

3.2

Generation of tester and driver representations

In order to perform effective hybridisations, the full-length ds-cDNA m ust be digested into smaller fragments, know n as representations. This is done using a four- base cutting restriction endonuclease that cleaves the DNA, on average, every 256 bp. One of two enzymes can be used; D pn II (New England Biolabs) - cutting at GATC (37°C), and Tsp 5091 (NEB) - cutting at AATT (65°C). In these RDA studies, D pn II was used.

To the 34 pi of full-length cDNA, 4 pi of D pn II buffer (lOx) and 2 pi of Dpn II w ere added and the sample w as incubated at 37°C for 3 hr.

The digest was diluted with 160 pi dH^O, and subjected to 2x p h enol/ chloroform and Ix chloroform extractions. 20 pi of NaOAc and 220 pi of isopropanol were added to the final aquous phase, and cDNA was precipitated at RT for 20 min. The pellet was w ashed w ith 70% ethanol, dried, and then resuspended in 10 pi dH^O (for every 4 PCR reactions that were perform ed above).

In order to prepare the tester representation for the hybridisation step, linkers m ust be added so that at later stages the tester DNA can be readily distinguished from the driver DNA. For historical reasons these linkers are term ed J-linkers, and comprise of a 24-mer and 12-mer. It is im portant to note that the 1 2-mer is specific for

use w ith a particular restriction enzyme, and m ust be changed if a different enzym e is used to cleave the cDNA.

J-24 5’-ACCGACGTCGACTATCCATGAACA

1-12 (Dpn II) 5 -GATCTGTTCATG (note: J-12 (Tsp) 5'-AATTTGTTCATG)

For both tester and driver representations (i.e. cut cDNA), 1 |xl and 0.5 pi w ere run on an ethidium brom ide stained 1.5% agarose gel. After a sufficient period, the intensity of the DNA "smears" was checked and recorded on an ImaGO machine (B&L systems). The smear of the driver was used this as a standard reference, taken as one unit (lU) of DNA. The volum e of tester w ould give a sm ear of equal intensity to 1 U of driver was estim ated and used to set up the following ligation: 1 U of tester cDNA, 12 pi ligase buffer 5x (GIBCO BRL), 6 pi of J-24 prim er (Ip g /p l), 3 pi of J-12 prim er

(Ip g /p l), dHgO to 57pl.

Primers were annealed in PCR machine by heating to 50°C then cooling l°C /m in to 15°C. Then, 3 p i ligase (G IB C O BRL) w ere added and the reaction m ixture was incubated overnight at 4°C.

3.3

First subtractive hybridisation

The following com ponents were mixed in a 0.5 ml eppendorf tube: 12 pi (0.2U) of ligated tester representation, 20 U of driver representation, and dH^O to 120 pi. After Ix phenol/chloroform and Ix chloroform extractions, the final aquous phase was collected and DNA was precipitated (on ice, 10-15 min) w ith 30 pi of 10 M NH^ acetate and 150 pi isopropanol. The pellet was w ashed twice w ith 70% EtOH. (The pellet should be glassy in appearance, it should not be big and white, which w ould imply too m uch salt in the procedure).

The pellet was resuspended in 4 pi of EEx3 buffer and transferred to a 0.2 ml thin- walled tube and cover w ith a drop of m ineral oil.

EEx3 buffer: 30 mM EPPS (Sigma), 3 mM EDTA pH 8.0.

The tube was placed in a PCR machine and incubated for 6 m in at 95°C. After

cooling to 72°C, 1 pi of 5M NaCl was added directly to the 4 pi of DNA w ithout rem oving tube from the block.

H ybridisation was prom oted by cooling 0.2°C every hour to 67°C, then 67°C for 24 hr, then cooling 0.2°C every hour to 63°C.

In the end, mineral oil was rem oved (as m uch as possible) and 156 |l i1 dH2 0 w ere

added by vigorous pipetting. Then four PCR reactions w ere set up as follows; 20 |il of hybridisation product, 5 pi PCR buffer (lOx), 1 pi dNTPs (lOmM) and 22 pi dH^O.

Tubes were incubated in the PCR block for 10 m in at 72°C (note - w ithout enzym e or primer). This m elted aw ay the J-12 prim er, which was not covalently linked to any fragm ent as it lacked a 5'-phosphate in the ligase reaction.

After 3 m in of the 10 m in incubation, 1 pi of Pic Taq polym erase (Cancer Research UK) w as added (note - this m ust be a Taq enzym e that does n o t need to be activated at 95°C before it is active). The Taq filled in the ends of hybridised DNA, m aking a com plem entary copy of the J-24 prim er which was ligated to the 5 -ends of the tester DNA.

After 8 m in of the 10 m in incubation, Ipl of J-24 prim er was added to each tube.

10 cycles of 1 m in 95°C and 3 m in 70°C followed.

The 4 PCR products were pooled and supplem ented w ith 200 pi dH^O and 1 pi (Ip g /p l) glycogen. After Ix phenol/chloroform and Ix chloroform extractions, 40 pi of NaOAc 2 M (pH 5.4) and 450 pi isopropanol were added to the final aquous phase. Precipitation occurred on ice for 20 min, and was followed by centrifugation (10 m in at 16,000xg).

The pellet w as resuspended in 34 pi dH^O. To this, 4 pi M ung Bean Nuclease Buffer lOx (G IB C O BRL) and 2 pi M ung Bean N uclease (20 U) (G IB C O BRL) w ere added, and the m ixture w as incubated at 30°C for 30 min. This digestion rem oved all single strand amplification products from the sample. To stop the reaction, 120 pi of Tiis-HCl 50 mM (pH 8.9) w ere added and incubated at 98°C for 5 min, followed by imm ediate transfer onto ice.

4 PCR reactions were set up as follows: 20 pi of sample (from above), 5 pi PCR buffer (lOx), 1 pi dNTPs (lOmM), 1 pi J-24 prim er, 22 pi H^O, 1 pi advantage 2 Taq polym erase (Clontech). These were subjected to 18 cycles of: 1 m in at 95°C, 3 m in at 70°C.

The 4 PCR products w ere pooled and 200 pi dH^O w ere added. After Ix phenol/chloroform and Ix chloroform extractions, the final aquous phase was

supplem ented w ith 40 |xl of NaOAc 2M (pH 5.4), and 450 pi isopropanol. DNA was precipitation on ice for 20 min, then centrifuged and the pellet w as resuspend in 40 pi (IH2O. This w as the first differential product (DPI), of which 4 pi and 2 pi w ere run

on a 1.5% agarose gel. lU of driver w as ru n next to the DPI to get a good estim ation of the concentration (U /pl) of DPI.

3.4

Second subtractive hybridisation

A second subtractive hybridisation involved the rem oval of J-linkers from DPI, allowing their substitution for N-linkers. The following digest was set up and incubated at 37°C for 3 hr: 2 U of D PI, 4 pi of Dpn II buffer (lOx), 2 pi of D pn II and dHgO to 40 pi.

To the digest, 160 pi d H 2 0 w ere added and 2x phenol/chloroform and Ix chloroform extractions were perform ed. The final aquous phase was supplem ented w ith 20 pi NaOAc 2 M, 1 pg glycogen and 220 pi isopropanol. After 20 m in at RT, the sam ple w as centrifuged (10 min at 16,000xg) and the pellet was w ashed with 70% EtOH, dried, and then resuspended in 20 pi dH 20.

0.2 U of digested DPI w ere mixed w ith 12 pi ligase buffer 5x (GIBCO BRL), 6 pi of

N-24 prim er (Ip g /p l), 3 pi of N-12 prim er (1 p g /p l) and d H 2 0 to 57pl. N -linkers w ere annealed in PCR machine by heating to 50°C and then cooling l°C /m in to 15°C. 3 pi ligase (GIBCO BRL) w ere then added and the reaction m ixture w as incubated overnight at 4°C.

N-24 5-AGGCAACTGTGCTATCCGAGGGAA

N-12 (Dpn II) 5 -GATCTTCCCTCG (note: N-12 (Tsp) 5 -AATTTTCCCTCG)

The second hybridisation (1:1,000 ratio) w as set up with: 6pl (0.02 U) of N-ligated

D P I, 20 U of driver representation, and H^O to 120pl. This m ixture w as subjected to Ix ph en ol/chloroform and Ix chloroform extractions, and DNA from the final aquous phase was precipitated w ith 30pl of am m onium acetate 10 M and 150 pi isopropanol (on ice, 10-15 min). The centrifuged pellet was w ashed twice w ith 70% ethanol, and

resuspended in 4pl of EEx3 buffer. After transfer to a 0.2ml thin-walled tube, the sample was covered with a drop of mineral oil and placed in a PCR machine, w here it w as incubated at 95°C for 6 min. After cooling to 72°C, Ipl NaCl 5 M w as added

directly to the 4pl of DNA w ithout rem oving tube from the block.

H ybridisation was prom oted by cooling 0.2°C every hour to 67°C, then 67°C for 24 hr, then cooling 0.2°C every hour to 63°C.

The second differential product (DP2) was generated identically to DPI, except in that the N-24 prim er now took the place of the J-24 prim er and the extension tem perature of the PCR reaction was 72°C rather than 70°C.

3.5

Isolation, sequencing and identification of