Definición de los frames y las variables

All solutions for use in work with RNA were prepared using autoclaved water that had been previously treated with 0.1% DEPC (DEPC water).

2.4.1

RNAE

H

C

L

Harvested cells were pelleted and resuspended in 0.5ml Trizol reagent (Invitrogen) and left to incubate at room temperature for 5 minutes. Then, 0.1ml of chloroform was added to each sample followed by 15 second of vigorous shaking. After a 3-minute incubation at room temperature, the samples were subjected to centrifugation at 12,000g for 15 minutes at 4°C. The supernatant was carefully removed and transferred to a fresh microcentrifuge tube, before 250µl isopropanol was added. The samples were gently mixed and incubated at room temperature for 10 minutes. The samples were then centrifuged at 12,000g for 10 minutes at 4°C and the RNA pellet was briefly dried before being resuspended in an appropriate volume of DEPC water. Samples were stored at -80°C. For large cell pellets, the volumes of all reagents used were doubled.

2.4.2R

T

The Superscript II Reverse Transcription kit (Invitrogen) was used to generate single stranded cDNA for use as a template in PCR reactions. An aliquot of 1µl of oligo dT primer (100mM stock) was added to 5-10ng of isolated RNA and was made up to 12µl total volume with DEPC treated water. The mix was incubated at 70°C for 10 minutes and then placed on ice whilst 1x First-Strand Reaction Buffer, dNTPs (0.5mM final concentration) and DTT (10mM final concentration) were added. The reaction was then incubated at 42°C for 5 minutes before adding 200U of Superscript II Reverse Transcriptase and continuing the incubation at 42°C for 50 minutes. Inactivation of the Superscript II Reverse Transcriptase was then achieved by heating to 70°C for 15 minutes. The cDNA was then stored at -80°C.

2.4.3

D

A

G

E

Denaturing agarose gel electrophoresis was used to separate RNA samples for subsequent northern blotting. Isolated RNA was subject to phenol/chloroform extraction to improve purity before measuring the concentration using the Nano-drop. A total of

5µg of isolated RNA was prepared in an 8µl total volume of DEPC water. Samples were then made up to a final volume of 20µl by the addition to final concentrations of 1x MOPS (40mM MOPS acid, 10mM NaOAc and 1mM EDTA pH 7.2), 35% formamide and 5.5% formaldehyde. Samples were then heated at 55°C for 15 minutes before adding 0.1µg/µl ethidium bromide and 1x loading dye.

Samples were then loaded and electrophoresed through the denaturing gel (1% agarose, 1x MOPS and 0.9% formaldehyde) in 1x MOPS buffer at 55V for 2 hours. The electrophoresis gel tank and all casting apparatus had been soaked with 3% H2O2 for 10

minutes at room temperature, rinsed in DEPC H2O and subsequently air dried prior to

gel preparation.

The electrophoresed RNA was visualised on a UV transilluminator to assess the migration, quality and loading before proceeding with northern blotting.

2.4.4N

B

P

G

RNA samples were electrophoresed as described above before being transferred to a Genescreen Plus membrane (Perkin Elmer). Transfer was carried out by capillary transfer in 10x SSPE (3.6M NaCl, 200mM phosphate buffer pH 7 and 20mM EDTA) overnight at room temperature. The membrane was then washed with 2x SSPE and visualised under UV light alongside the gel to confirm transfer efficiency, and rRNA migration. The membrane was then baked at 80°C for 2 hours in a vacuum drier. The membrane was blocked for 2 hours at 42°C in 10ml pre-hybridisation solution (50% formamide, 5x SSPE, 1% SDS and 5x Denhardt’s solution) before adding any probes. Various 32_{P-labelled DNA probes were produced to target specific desired RNA}

sequences. DNA templates used for these probes were produced by PCR. These DNA templates (100ng) were denatured in 9µl DEPC water at 95°C for 4 minutes before cooling and adding 1x random hexamer mix, 5U Klenow fragment (DNA polymerase I, Promega) and 2µl 32P α-dCTP (Perkin Elmer NEG513H) in a 15µl reaction. The reaction mix was then incubated for 1 hour at 37°C and then passed through Illustra G- 25 columns (GE Healthcare) according to manufacturer’s guidelines to remove free nucleotides. The activity of the final probes was then estimated using a Cerenkov counter and a volume equivalent to approximately 500,000cps was added to the pre- hybridisation solution after the aforementioned membrane-blocking step. The

membrane was then hybridised with probe by incubation in this solution overnight at 42°C with rotation.

After hybridisation, the membrane was washed twice in 2x SSPE for 15 minutes at room temperature before a third wash in pre-warmed 2x SSPE/2% SDS and incubated at 65°C for 15 minutes.

The membrane was then wrapped in saran wrap and exposed to a PhosphoImage screen in a cassette before visualising the signal using the Typhoon FLA 9500 and ImageQuant software (GE Healthcare).

For subsequent hybridisation of probes to other targets, the membrane was washed twice in 2x SSPE and once with 2x SSPE/2%SDS at 65°C as described above before adding the next probe. If two different probes targeted RNAs with similar molecular weights then it was necessary to strip the membrane of the first signal before re-probing with the second probe. Membrane stripping was performed by washing the membrane twice in boiling 0.1x SSC (1x SSC: 150mM NaCl, 15mM sodium citrate) and once with SSC/0.1% SDS for 15 minutes. Any radioactive signal present on the membrane was assessed using a Geiger counter to ensure the removal of the previous signal before hybridising with another probe as described above.