1) Remove organs from six C57Bl mice (do cervical dislocation immediately before organ removal) and freeze directly into liquid nitrogen. Store at -70°C. For lacrimal gland, in which mRNA yields directly from the organ have often been low, a better approach is to do an isolation of lacrimal acinar cells from six C57Bl mice (see method in Lab Protocols; leave out Percoll step) first, then extract mRNA. To extract from cultured cells (ie. HT1080), begin with one T75 flask (approx. 2 x 106 cells).
2) Have Pharmacia kit reagents warmed to RT for 30 min. Check if Extraction buffer is in solution; if not warm to 37°C then cool to RT. Have water bath at 65°C and 0.5 ml/purification of elution buffer warming at 65°C. Resuspend Oligo(dT) and pipet 1 ml aliquots into individual microcentrifugation tubes (1 ml/purification). Weigh out 0.1 - 0.23 gm of tissue, and place in a 7 ml mortar (alternatively, can use a polytron). Add 0.4 ml of extraction buffer; use pestle to homogenize. Add 0.8 ml of elution buffer, mix, homogenize and transfer to a clean 1.5 ml microcentrifugation tube. Centrifuge for 1 min (RT) full speed. Centrifuge Oligo(dT) for 2 sec (just before gets to full speed, release button; spinning longer makes resuspension of Oligo(dT) difficult). Discard supernatant from Oligo(dT) and add to it 1 ml of supernatant from tissue homogenization. Mix for 3 min by inverting on rotor.
3) Spin 2 sec, discard supernatant. Wash five times each with 1 ml of High Salt Buffer (quick spins between several inversions to mix). Wash two times each with 1 ml of Low Salt Buffer. Resuspend in 0.3 ml of Low Salt Buffer and transfer to a Microspin Column (break off end before use) in a microcentrifuge tube. Spin at full speed for 5 sec. Discard effluent and add 0.5 ml of Low Salt Buffer. Spin and wash two more times with 0.5 ml each of Low Salt Buffer. To elute, place column in a fresh microcentrifuge tube and add 0.2 ml of prewarmed Elution buffer. Spin at full speed for 5 sec. Add a second 0.2 ml of prewarmed Elution buffer and spin again. Place eluted mRNA on ice.
4) To determine OD260 of mRNA, have a quartz cuvette soaking in conc. HCl/methanol (1:1) for 1 hr prior to use to remove RNase. Rinse well with DEPC treated ddH2O. Zero spectrophotometer with elution buffer then add all of mRNA sample to cuvette to determine OD260 and OD260/OD280. For µg/ml RNA, multiply OD260 value times 40 (1 OD260 is 40 µg/ml). For storage, precipitate mRNA with 10 µl of Glycogen solution, 40 µl of potassium acetate solution and 1 ml of -20°C 95% EtOH. Store at -70°C.
5) For Northern analysis, have heating block at 56°C and pour denaturing gel (mid-size gel: to 0.8 gm of agarose, add 78.4 ddH2O and 5 ml of 20 x E buffer; heat to dissolve; let cool to not too hot to touch, then quickly add 16.6 ml of 37% formaldehyde, swirl to thoroughly mix and pour). Spin precipitated mRNA for 15 min (4°C, full speed). Carefully remove and discard supernatant. Wash pellet with RNase-free 80% EtOH, dry and make up in 20 µl of 'Buffer A'. Also have size standards including an RNA ladder (mix 20 µl of ladder + 28.5 µl of 'Sample Buffer') and 28S/18S rRNA standard (mix 20 µl of ladder + 28.5 µl of 'Sample Buffer'). Heat at 56°C for 15 min, then place on ice. Add 4.9 µl of 'Glycerol Stain' to mRNA and 12.5 µl to standards. Load onto gel immersed in 1 x E/formaldehyde buffer (see below) with standards in the outside lanes. Run overnight at 35 V (constant).
20 X E Buffer
Na2HPO4.7H2O 32.72 gm
NaH2PO4.H2O 10.85 gm
ddH2O to 1000 ml
1 x E/Formaldehyde Buffer
ddH2O 773 ml
20 x E buffer 50 ml
37% formald. 177 ml
formamide 145.2 µl
20 x E buffer 12.8 µl
37% formald. 42 µl
DEPC ddH2O 118 µl
sample buffer 200 µl
DEPC ddH2O 7 ml
glycerol 3 ml
bromophenol bl. 25 mg
xylene phenol 25 mg
Add 0.8 ml of DEPC (Sigma #D-5758) to 800 ml ddH2O. Warm overnight at 37° then autoclave.
6) Carefully cut out the standards lanes and place in ethidium bromide (5 µg/ml; 5 µl of stock per 100 ml of ddH2O) for 30 min. Wash overnight in ddH2O, then photograph on transilluminator with florescent ruler. The rest of the gel (cut off the left bottom corner to aid in orientation) is transferred to a clean glass tray and immediately denatured in 900 ml of 50 mM NaOH/10 mM NaCl (to 893.7 ml of ddH2O add 4.5 ml of 10 N NaOH and 1.8 ml of 5 M NaCl) for 20 min on a rotator. Carefully pour off and add 900 ml of 0.1 M Tris, pH 7.6 to neutralize (20 min on rotator). Pour off and add 900 ml of 20 x SSC for 45 min with gentle rotation. To keep gel immersed during above steps, can balance an inverted ddH2O filled 50 ml conical on it, making sure that the top surface of the gel is wet before placing on the conical.
7) During the above 20 and 45 min steps, preparations for the overnight transfer can be initiated. Needed are: (i) a glass tray or lucite box filled to a depth of 2 cm with 10 x SSC, (ii) a lucite support for the gel (can invert the gel mold in the reservoir), (iii) a long piece of 3 MM paper which stretches over the lucite gel support on either side into the 10 x SSC; wet with 10 x SSC, (iv) nitrocellulose (Amersham Hybond ECL) cut to the same size as the gel (wear gloves in this and all steps), (v) two pieces of 3MM paper cut to the same size as the gel; place one on the long 3 MM piece and wet with 10 x SSC, (vi) a 6-8 inch stack of paper towels cut to the same size as the gel, (vii) a glass tray containing ddH2O, (viii) a glass tray containing 10 x SSC, (ix) two 10Ó x 12Ó pieces of X-ray film, and (x) flat forceps.
8) To set up the transfer, sandwich the gel between the X-ray film pieces and carefully invert (do over the 20 x SSC). Place back side up on the wetted gel-size 3MM paper (on inverted mold). Remove any air bubbles by gently rolling a capless 50 ml conical over the gel. Add some 10 X SSC to keep from drying out. Completely surround the gel with Parafilm or plastic wrap. Next, wet the nitrocellulose paper by floating on, then immersing in, the ddH2O. Immerse the wet filter in 10 x SSC for 5 min. Holding the filter by two sides, carefully set down exactly on the gel (canÕt reposition). Carefully roll the capless conical over the filter to ensure removal of any air bubbles. Add the remaining gel-size piece of 3MM paper (wet with 10 x SSC) and roll with conical. Carefully add the cut paper towels to a height of 6Ó. Balance a glass tray on top of the towels to weigh down; stabilize the tray with tape running down on all sides to the 10 x SSC reservoir. Cover with plastic to reduce loss to evaporation. If transfer is initiated in the morning, carefully change the paper towels (can reuse the top dry ones) before leaving at the end of the day. Let the transfer go overnight.
9) Have 80°C vacuum oven heating up. Remove the paper towels and, with the gel/filter still in place, invert. Use an indelible fine tip marker to mark on the filter the edges of the gel (including the cut corner) and the loading wells. Record experimental number at the top. Peel off the gel and stain for 45 min in 0.5 µg/ml ethidium bromide (then place in ddH2O and examine/photograph on transilluminator). Place filter in 6 x SSC for 5 min to wash, then place on 3MM paper and dry for at least 30 min. Place the dried filter between two pieces of 3MM paper and bake under vacuum for 1-2 hr.
10) To carry out hybridization to filter, prehybridize filter for 4 hr at 42°C (in prehyb/hyb. plastic boxes; gently rotation) in a prewarmed solution of 30% formamide, 5 x SSPE, 0.1% SDS, 5 x DenhardtÕs, 0.2 mg/ml sonicated denatured salmon sperm DNA.
Prehybridization SolÕn Hybridization SolÕn ddH2O 15.3 ml ddH2O 15.3 ml Formamide 15 ml Formamide 15 ml 20 x SSPE 12.5 ml 20 x SSPE 12.5 ml 20% SDS 0.25 ml 20% SDS 0.25 ml 50 x DenhardtÕs 5 ml 50 x DenhardtÕs 5 ml Salmon Sp. DNA 2 ml Salmon Sp. DNA 2 ml Poly A 5 µl Poly A 5 µl 32cDNA probe 50-100 µl
(note: Salmon Sperm DNA stock is 5 mg/ml; Poly A stock is 10 mg/ml; make up prehyb and hyb sol'ns in 50 ml conicals)
11) During prehybridization, label cDNA probe (purified insert) by random priming method (Pharmacia #27-9251-01). Reconstitute reaction mix by adding 20 µl of ddH2O and place on ice for 5-60 min. Make 1.8 µl of insert up in 25 µl of TE, pH 8 and denature for 3 min at 95°C. Cool on ice for 2 min, then add diluted/denatured insert to reaction mix. Behind lucite, add 5 µl of 32PdCTP (ICN #33004X). Carefully mix by pipetting up and down. Incubate at 37°C for 15-30 min. Purify probe using a Nick Spin Column (Pharmacia #17-0862-02) or equivalent (Qiagen #28304) and check cpmÕs in Scintillation counter. Denature probe (3 min at 95°C, on ice for 2 min) prior to addition to hybridization solution. Hybridize overnight at 42°C with gently rotation.
12) Remove filter into 400 ml of room temperature 2 x SSC, 0.1% SDS (40 ml of 20 x SSC and 4 ml of 20% SDS made up to 800 ml in ddH2O). Wash for 5 min on rotator. Do two additional washes. Transfer filter to 400 ml of prewarmed (42 - 65°C; use 42°C for wheat-mouse and 65°C for mouse-mouse hybridizations) 0.1 x SSC, 0.1% SDS (4 ml of 20 x SSC and 4 ml of 20% SDS made up to 800 ml in ddH2O). Wash for 20 min on rotator. Do a total of three 20 min washes.
13) Allow filter to dry on fresh bench paper, then place on Whatmann 3MM paper in a large x-ray cassette holder. Cover with plastic wrap. Under red light in the dark room, place x-ray film on, followed by an enhancing screen. Close up and expose overnight at -70°C.
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