RNA manipulation

a)Viral RNA extraction from tissue culture

A sub-confluent monolayer of BHK-21 seeded in 6-wells plate cells was infected at an MOI of 1. Two-hundred µl of inoculum (virus stock in PBS - 2% NCS) was used and cells were incubated for 1h at 37°C and rocked every 20 min. Two ml of media were added to the cells and then incubated at 33°C. The supernatant was removed 48h post-infection.

(i)Use of Qiagen kit

The cell monolayer was lysed using 600 µl of RLT buffer and the RNA was extracted from the lysate using RNeasy Mini Kit (Qiagen). Samples were processed using the Qiacube according to the manufacturer protocol.

(ii)Trizol extraction

The cell monolayer was lysed using 500 µl of Trizol Reagent (Invitrogen) for 10 min at RT. Samples were collected in a 2 ml screw cap tube and can be stored at -80 C to be used later. If starting from a frozen sample, tubes were left on the bench until completely thawed, 100 µl of chloroform was added. The different phases were mixed by inverting the tube 4 to 6

87 times. Samples were incubated at RT for 3 min and mixed before spinning at 12 000 rpm for 15 min at 4°C. The aqueous phase was transferred to a new tube and 75% (v/v) of isopropanol was added and mixed. Samples were incubated for 15 min at RT before being centrifugated at 12 000 rpm for 20 min at 4°C. The supernatant was poured off and 750 µl of 75% EtOH added. Samples can be stored at this point at -20°C if needed later. Samples were centrifugated at 7 500 rpm for 5 min at 4°C, the supernatant poured off and the pellet air-dried. Pellets were resuspended in 30 µl of RNase-free water.

(iii)Quantification

To quantify the amount of RNA present in each sample, a 2 µl drop was used on a spectrophotometer, Nanodrop (Thermo Scientitist).

b)

Reverse Tanscription – PCR

To produce cDNA from RNA, 1 µg of total cellular RNA was diluted in water to a final

volume of 12 µL, 1 µL of an appropriate primer was added. The mix was incubated at 65°C

for 5 min. After cooling on ice, 4 µL M-MLV 5x buffer, 1 µL dNTPs 10 mM and 1µL RNasin

were added and the mix was incubated at 42°C for 2 min. Then, 1 µL M-MLV Reverse

Trancriptase was added and the final reaction mix was incubated at 42°C for 2.5h. All incubation periods were performed in a thermocycler and all reagents were from Promega.

c)Northern blot

(i)Preparation of the gel

1.2 g of agarose were added to 100 ml 1X TAE and bring to the boil. The electrophoresis tank, the tray (11 x 14 cm), the comb and the stop-wedges were cleaned with RNaseZAP before pouring the gel. The gel was left to set for 1h.

(ii)Preparation of RNA samples

3 µg of RNA was prepared in 25 µl formamide, 2.5 µl RNA loading dye, 0.1 µl ethidium bromide and deionised water up to a final volume of 30 µl per well. The ladder was composed of 10 µl RiboRuler High Range RNA Ladder (Fermentas), 10 µl of 2X RNA Ladder (Fermentas) and 10 µl deionised water.

Samples and ladder were heated at 70ºC for 5min, chilled on ice for 5min and loaded on the gel.

Chapter 2: Materials and methods Methods

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(iii)Electrophoresis and blotting

The gel was run in 1X TAE at 75 V for 3 to 4 hours or until the dye reached a 0.5 cm distance from the bottom of the gel.

After electrophoresis, a picture of the gel was taken in a UV transilluminator then the gel was washed twice in 10X SSC for 5 min under constant rocking at RT. At the same time the positively charge Hybond-N+ membrane was equilibrated in 10X SSC.

The gel running tray (11x14 cm) was placed upside down on a shallow tray. A stack was formed on top of the gel running tray and was composed from bottom to top by: 8 cm of dry blotting pads (11x14 cm, VWC), 3 mm chromatography paper (11x14 cm) soaked in 10X SSC (11x14 cm, Whatman), the equilibrated membrane, the equilibrated gel (wells up) and 3 mm chromatography paper (11x14 cm, Whatman) soaked in 10X SSC. Bubbles were pressed out of the stack using a 10 ml pipette. Two long Whatman pads (14x40 cm) were soaked in 10X SSC and placed on top of the stack so that both ends reached the swallow tray. Two 10 ml pipettes were placed on each side of the stack to prevent the long Whatman from touching the stack. The shallow tray was filled with 10X SSC. A plastic board and a 1 kg weight were place on top of the stack. The apparatus was wrapped in cling film and left over night to allow the capillary-driven transfer of RNA from gel to membrane.

(iv)UV cross-linking of the membrane

Once the blotting is complete, the position of the wells was marked using a ball point pen. The membrane was removed, rinsed in 2X SSC for 5min and hung to dry. The membrane was place RNA-side down on a transilluminator and the RNA was cross-linked to the membrane by UV (302 nm) for 3 min. The membrane was stored at 4°C until used for hybridization.

(v)Hybridisation of DIG-labelled probes

The lane containing the ladder was cut off and stained with a solution of 1X Methylen Blue.

The membrane was placed in a hybridization tube with 4 or 10 ml (for a small or large tube respectively) of 50% formamide pre-hybridisation buffer (warmed to 68ºC) for 30 min under constant rotation at 68ºC. One-hundred and fifty ng of digoxigenin labelled probes were denatured by adding them to 100 µl deionised water, and then boiled for 5 min. After cooling on ice for 5 min, the probes were spin down briefly. The pre hybridisation buffer was poured off and replaced by 4 or 10 ml 50% formamide hybridisation buffer containing the probes. The hybridization tubes were rotated over-night at 68ºC.

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(vi)Washes and detection

All further steps were carried out in a hybridisation tube, under constant rotation and, if not mentioned, at RT. The membrane was washed under increased stringency as follow:

twice in 2X SSC - 0.1% SDS for 15 min

twice in 0.1X SSC – 0.1% SDS for 30 min at 68ºC

in washing buffer for 5 min

The washing buffer was replaced by 10 ml of 1X Blocking buffer. After 60 min, the Blocking buffer was poured off and 10 ml antibody buffer was added for 60 min. Then, the membrane was washed twice in 10 ml washing buffer before being incubated in 10 ml detection buffer for 5 min.

The membrane was removed from the tube and placed on a translucent plastic sheet. A few drops of CDP-Star solution were dropped onto the membrane and the plastic was folded over it. Bubbles were removed to form a uniform layer of liquid. CDP-Star was left for 5 min before the excess was rolled out. The membrane was then exposed to X-Ray film for up to 30 min.