2.5.1.1
Human blots
Hum an 12-lane m ultiple tissue blots and brain II, III and IV blots w ere purchased from BD Biosciences Clontech.
2.5.1.2
COS-7 and Xenopus oocyte blot
2.5.1.2.1 RNA purification
RN A from CO S-7 cells and Xenopus oocytes was isolated using the R N A zol B Phenol- chloroform extraction m ethod (GibcoBRL). COS-7 cells w ere cultured to confluence in a T75 tissue culture flask (see section 2.5.3.1.1), washed tw ice w ith sterile, cold PBS and 4ml o f RN Azol B reagent added to the flask. Cells were w ashed from flask and 1ml placed into four sterile 1.5 ml tubes. A pproxim ately 25 stage V and V I oocytes were placed in a 1.5ml tube and any N D 96 buffer (see section 2.6.1) draw n off. 1ml o f RNAzol B was added to each tube. Cells were hom ogenised using a glass-teflon hom ogeniser.
200|
l l1
o f chloroform was added per 2ml o f hom ogenate and tubes were shaken vigorously for 15 seconds and then placed on ice for 5 min. The suspension was centrifuged at 12,000 x g at 4°C for 15 min. The upper aqueous phase was draw n o ff and placed in a fresh tube. An equal volum e o f isopropanol was added and sam ples w ere stored at 4°C for 15 min. Samples were centrifuged at 12,000 x g at 4°C for 15 min. The RN A form ed a white yellow pellet at the base o f the tube. O ptional washes were perform ed until the pellet was almost com pletely w hite (especially required forC hapter 2 M aterials & M ethods
oocyte RNA). These proceeded as follows: The pellet was re-suspended in 500|il dH iO , and 50|il N aA cetate and 500|li1 Phenol:C hloroform :isoam ylalcohol (25:24:1) added. The tube was vortexed and centrifuged at 12,000 x g at 4°C for 5 min. The aqueous phase was removed, 1ml absolute ethanol added and the sam ples stored at - 20°C for 30 min. Samples were spun at 12,000 x g at 4°C for 15 m in. Ethanol was rem oved and the tubes spun for a further 2 min. Excess ethanol was draw n o ff and the pellet air-dried for 5 min. Pellet was re-suspended in 50)il D EPC dH 20.
2.5.1.2.2 Blot creation
A denaturing agarose gel was cast (see Table 2.13 for constituents).
Quantit)
dHiO 152.25 ml
Agarose 2.45 g
10 X MOPS 17.5 ml
Formaldehyde 5.25 ml
Table 2.13 - Constituents of a IxMOPS denaturing agarose gel
Agarose and dH20 was warmed until the agarose has dissolved, then allowed to cool to approximately 60°C. The MOPS and formaldehyde was added at this point and the gel allowed to set.
RNA samples w ere prepared as in Table 2.14.
Table 2.14 - RNA sample preparation for denaturing gel
Samples were heated to 55°C and placed on ice and 0.5|il EtBr and 5pi gel dye added.
Reagent Quantity RNA 20 pg 10 X MOPS 5 pi Formaldehyde 8.75 pi Deionised formamide 2.5 pi dHzO To 50 pi
Samples were loaded onto the gel and run overnight at 20m V (in 1 X M O PS running buffer). The gel was then w ashed in 1 x TA E for 30 min and a nitrocellulose filter cut to the size o f the gel, in 1 x TAE for 10 min. RNA was transferred onto the filter m em brane by overnight electroblotting (Figure 2.7) run at 0.7 - 0.8 m A in 1 x TAE. RN A was cross-linked to the m em brane using an ultraviolet stratalinker (Stratagene) set on auto cross-link (120 000 pjoules / cm^). Blots were stored in an air-tight bag at -20°C until ready for use.
Chapter 2 Materials & Methods
Figure 2.7 - A pparatus for tran sfer of RNA from d en atu rin g gel to nitrocellulose m em brane using an electroblotter
Biol performed overnight in I x TAE at 0.7-0.8mA. -t- and -
denote anode and cathode
respectively. F o a m p a d W h a t m a n f i l t e r s ( x 3 ) N i t r o c e l l u l o s e f i l t e r G e l W h a t m a n f i l t e r s ( x 3 ) F o a m p a d
2.5.1.3 Probe design and synthesis
2.5.1.3.1 cDNA probes
The y subunit northern probe templates were synthesised from lOOng o f plasmid containing the appropriate complete y subunit cDNA. Pfu Turbo parameters were employed to amplify fragments using the primer pairs detailed in Table 2.15, and the products o f the PCR reactions purified by agarose gel electrophoresis and gel extraction. The quantity o f probe synthesised was estimated by running a sample of the purified probe template on a high percentage agarose gel against a low mass DN A ladder (GibcoBRL).
Probe P rim er S trand P rim er Sequence y subunit
cDNA location Probe size Y2 FM24 FM42 Coding Complement 5 ’ -C ATGTTTATCG ACCGGC-3 ’
5 ’ -TGTAC ATGG AG ATCTCCG-3 ’ nt 597-814 218nt
FM26 FM27 Coding Complement 5 ’ -C AG A A ATTGTAGG AGTGGTT-3 ’ 5 ’-GGTG A AC ATCG AG ATGTCAG-3 ’ nt 560-792 233nt Y4 FM28 FM29 Coding Complement 5 ’-GGGCGTCCTGGCTGTAA A-3 ’ 5 ’-GGCCCCTGTG ATCTTCAG-3 ’ nt 594-804 21 Int
FMI 20 Coding 5 ’ -GGCGTG ATGTCCGTGTACCT-3 ’
Y7 FM121/
FM50 Complement 5 ’-TC ATTTGG ATGG AC ACGTCG-3 ’
nt 577-763 187nt Burgess Y? FM142 FMI 43 Coding Complement
5 ’ -CGGTC AGC ACTG ACTACTGGC-3 ’
5 ’ - ATG ACCTCGTCGTTG ATGC-3 ’ nt 80-482 403nt
Table 2.15 - y subunit n o rth ern probe prim ers
Stripable [a-^"P] radio-labelled y subunit cDN A probes were assembled according to the Strip-EZ DN A Probe Synthesis and Removal Kit (Ambion, Abingdon, UK). 25ng
Chapter 2__________________________________________________M aterials & M ethods
linearised tem plate was placed in a tube, made up to 9]x\ with TE buffer and denatured by heating at 95-100°C in a hot block for 5 min. The sam ple was snap frozen on dry ice, thawed, m icrfocentrifuged and place on ice. To the denatured D N A w as added: 2.5|il 10 X decam er solution, 5p.l 5 x buffer -dA T P /cC T P , 2.5)il 10 x dCTP, 5.0p.l [a-^^P]
dA TP (3000 Ci/m m ol (370M Bq/ml), Am ersham , AA0004), 1.0 p,l exonuclease-free K lenow fragment. Contents were gently m ixed and incubated at 37°C for 30 m inutes. The reaction was term inated by adding IjLil 0.5M EDTA. U nincorporated nucleotides were rem oved by passing the synthesis reaction through a C hrom aspin-10 colum n (BD Biosciences Clontech). This rem oved >90% of products <4bp and dN TPs, and recovers >50% lObp, >70% 15bp and >90% o f >30bp fragments. Once storage buffer had been spun out o f the colum n and a sem i-dry colum n matrix established, the sam ple was applied to the centre o f the gel b ed ’s flat surface. The colum n was placed in a 1.5 ml tube and spun at 700 x g for 5 min. The purified sample was collected in the tube.
27|il o f the purified probe was transferred to a fresh tube and diluted ten-fold in lOmM ED TA (243 p.1). The diluted probe was incubated at 90°C for 10 min. T he probe was transferred directly to a container containing a pre-hybridised blot (see section 2.5.1.4).
2.5.1.3.2 O ligonucleotide probes
A 49m er oligonucleotide probe FM 98, 5 ’-G C TG G G TG TTC A TG G G C A TC A C A TA T- T C T A TG G TG A A G CA A CG CCCC CG -3 ’ was designed to the com plementary sequence o f nt 349-397 o f the com plete CACNG 5 in silico prediction, a region that did not cross any exon-intron boundaries. lOpmol o f the probe was labelled with [y-'^^P] dATP according to the K inaseM ax 5 ’ End-Labeling Kit (Am bion) forw ard reaction protocol. The T4 polynucleotide kinase catalyses the transfer of the y phosphate from [y-^^P] dA TP to the 5 ’ -O H o f the oligonucleotide. 10 pmol o f the FM 98 oligonucleotide was incubated with 21 pm ol [y-'^^P] dATP (7000, Ci/m m ol, 150m Ci/m l), 2p.l 5 x forw ard buffer and l|il T4 polynucleotide kinase (10 U/)l11) in a final volum e o f 10 p.1 at 37°C for 30 min and then the reaction halted by a 2 min 95°C incubation.
Chapter 2__________________________________________________M aterials & M ethods
2.5.1 A
Hybridisation and washing
2.5.1.4.1 Blot pre-hybridisation
Before every hybridisation blots were pre-hybridised for Ih in ExpressH yb solution (BD B ioscience Clontech).
2.5.1.4.2 Hybridisation
Blots hybridised with cD N A radio-labelled probes were incubated overnight at 65°C in ExpressH yb solution. Blots hybridised with oligonucleotide radio-labelled probes were incubated overnight at 37°C in ExpressH yb solution.
2.5.1.4.3 W ashes
High stringency washes for cD N A probe hybridised blots w ere perform ed as follows: ExpressH yb and probe was poured off from container and blots were w ashed briefly in solution 1 (Table 2.16) at 65°C. W ash solution was poured o ff and the blot w ashed again in solution 1 for 10 min at 65°C. Blots were transferred to a tray containing solution 2 at 65°C and washed w hilst shaking slow ly on a rotary shaker for 10 min. Blots were rem oved, em issions checked and re-w ashed in solution 2 (Table 2.16) until a suitable reading obtained (just above background).
Solution 1
Solution 2
Table 2.16 - Northern blot wash solutions2 X SSC O . l x S S C s s c = Saline Sodium Citrate, SDS = Sodium dodecylsulphate.
0.05 % SDS 0.1 % SDS Solutions made fresh in 1 1 volumes in dH2 0.
Low Stringency w ashing of oligonucleotide probe hybridised blots w ere perform ed as follows: Blots w ere w ashed for 30-40 min in solution 1 at 37°C with changes o f the solution every 10 min. Blots w ere then w ashed in solution 2 at room tem perature for 40 min with continuous shaking and one change o f solution after 20 min.
Chapter 2__________________________________________________M aterials & M ethods
2.5.1.5
Exposure, doveloping and image processing
Blots were w rapped in cling-film and placed dow n on X -O M A T blue film (Kodak) in an auto-cassette. The cassette was stored at -80°C for 1-4 days, or until a suitable exposure was obtained. Films were developed and fixed using and autom atic X -O M A T processor (Kodak) or sim ilar device. D eveloped films were scanned into a PC and im ages processed using the A dobe Photoshop versions 4.0 & 5.5 softw are (Adobe System s, London, UK). In all circum stances, no attem pt was m ade to quantify the intensity of probe signal, although relative transcript detection levels could be com pared on the sam e blots.