Isolation of total RNA from biopsy samples and cell cultures
The Qiagen R N A /D N A buffer kit and Qiagen-tip 100 was used to extract RNA from biopsy sam ples and short term cell cultures.
Preparation of cell lysate
Three confluent 150cm^ tissue culture flasks of cells (3 1 x 10^) w ere
required. The m edia was aspirated from the flasks and the cells w ash ed w ith 10ml HBSS before 4ml of trypsin was added. The cells w ere incubated at 37°C un til they had detached from the bottom of the flasks at w h ic h point 6ml of PBS w as added to the cells and the total volum e transferred to a 30ml universal. The cells were pelleted by centrifugation lOOOrpm for 5 m inutes. The su p ern atan t was aspirated and 2ml of buffer QRLl added to the cell pellet. The lysate was then passed 3 or 4 tim es th ro u g h an 18 gauge needle to hom ogenise the cells w hich were then transferred to a 50m l polypropylene tube.
Preparation of tissue lysate
Fresh tissue or thaw ed biopsy tissue (^25mg) was finally chopped w ith crossed scalpel blades (size 10) before being transferred to a 50m l polypropylene tube containing 2ml of buffer QRLl. The tissue was hom ogenised by draw ing it through an 18 gauge needle 3 or 4 times.
RNA isolation
The lysate was m ixed w ith 2ml of buffer QRVl and centrifuged for 20 m inutes at 3000rpm. The supernatant was carefully decanted into a sterile 15ml polypropylene centrifuge tube (Helena Biosciences) and 3.2ml of ice- cold isopropanol added. The tube was then incubated on ice for 5 m in u te s before being centrifuged at 3000rpm for 30 m inutes. M eanw hile 3ml of buffer QRE was pipetted onto a Qiagen tip 100 to equilibrate it. The c o lu m n
the pellet resuspended in 1ml of buffer QRLl. The pellet w as v o rtexed u n til it h ad dissolved. The sam ple was m ixed w ith 9ml of buffer QRV2 and pipetted on to the Qiagen tip. The sam ple was allow ed to enter th e resin by gravity flow before 12ml of buffer QRW w as added to the tip. W hen the buffer had passed though the tip w as placed over a clean 15m l polypropylene centrifuge tube and 6ml of preheated (75°C) buffer QRU was ad d ed to the tip. The RNA was eluted by gravity flow. One volum e of ice- cold isopropanol was added to the elute and incubated for 10 m inutes o n ice before being centrifuged for 30 m inutes at 15,000 x g i n order to precipitate the RNA. The supernatant was discarded and 5ml of 70% ethanol added to the pellet. The sam ple was vortexed and centrifuged at SOOOrpm for 15 m inutes. The supernatant was rem oved and this step repeated. The RNA pellet was air dried for approxim ately 10 m in u te s before being resuspended in 200^1 of DEPC w ater and pipetted into a 1.5ml
polypropylene tube for storage at -70°C. Each RNA extract was
electrophoresed on a sodium phosphate gel to determ ine its quality.
Agarose gel analysis of RNA
All electrophoresis equipm ent w as cleaned w ith detergent solution, rin sed in w ater, dried w ith ethanol and then filled/covered w ith a 3% solution of hydrogen peroxide. After 10 m inutes at room tem perature the e q u ip m e n t w as then rinsed thoroughly w ith DEPC treated water.
A 1% agarose gel w as prepared using IX sodium phosphate buffer. T en m icrolitres of each RNA sam ple w as electrophoresed for 1 h o u r at lOOV in IX sodium phosphate buffer containing 30jul lO m g/m l eth id iu m brom ide. A 0.24-9.5Kb RNA ladder (Invitrogen Ltd) w as used to determ ine the size
of the RNA sample. Products were visualized using a U V
tra n sillu m in a to r.
Expression of M D R l gene in ependym om as u sin g RT-PCR
The Q iagen OneStep RT-PCR kit was used to investigate the expression of the M D R l gene in RNA sam ples from paediatric ependym om as. T he
protocol for the Qiagen OneStep RT-PCR kit allows reverse tran scrip tio n and PCR amplification to be carried out sequentially in the sam e tube. T h e prim er sequences for each prim er used are detailed in Table 2.3. RNA was extracted from a sam ple of norm al h u m an brain tissue, as p rev io u sly described, taken during routine operation and used as a control in all RT- PCR am plifications. A negative control was included w ith all reactions, w hich consisted of a reaction containing no tem plate RNA. N o p ro d u ct should be detected after am plification unless there has been cross contam ination d uring the set-up of the reaction.
Primer Name Primer Sequence Size Reference
MDRIF F R
5' GGG ACC GCA ATG GAG GAG 3' 5' GAG ATT CAT GAA GAA CGC TG 3'
308bp (Sugawara, Watanabe et al, 1992)
MDRIE F R
5' GGG ACC GCA ATG GAG GAG 3' 5' TCC AGC CGC ATG GAT GA 3'
179bp (Sugawara, Watanabe et al, 1992) P2MG F
R
5' ACC CGC ACT GAA AAA GAT GA 3'
5' ATC TTC AAA GCT CCA TGA TG 3'
120bp (Noonan, Beck et al, 1990)
T able 2.3: M DRl prim er inform ation
PCR am plifications w ere carried out in a total volum e of 50jul co n tain in g 5jul of RNA solution. The reaction mix consisted of 400jum of each dNTP, 0.6/im of each forw ard and reverse prim er, lOjul of 5 X RT-PCR buffer
containing 12.5mM MgCl2 and 10^1 5 X Q-solution. For rev erse
transcription the sam ples were incubated at 50°C for 30 m inutes. For PCR am plification the sam ples were heated at 95°C for 15 m inutes on a th e rm a l cycler followed by 35 cycles of dénaturation at 94°C for 1 m inute, a n n ea lin g at 55°C for 1 m inute and extension at 72°C for 1 m inute. A final ex ten sio n step consisted of incubation at 72°C for 10 minutes.
After am plification lOjul of each sample was electrophoresed on a 2% agarose gel containing 5^1 lO m g/m l ethidium brom ide for 1 h o u r at lOOv
the PCR products. Images of the gel were captured using the G eneG enius Bio-imaging system and Gene snap software (Syngene, Cambridge, UK). Photographs of each gel were also taken using a polaroid direct screen instant cam era fitted w ith polaroid black and w hite film. M D R l m R N A was judged to be present in the tu m our sam ple if a band could be clearly seen on the gel image. If no band was present for M D R l b u t a ban d could
be seen for p2MG, that tu m o u r was judged not to express M D R l .
Expression w as graded according to band intensity: +++ to sam ples w ith a strong band, ++ to sam ples w ith an obvious band and + to sam ples w ith a faint positive band.