RNA实验方法

Solublization of RNA in Formamide

contributed by James McCaughern-Carucci, Yale Universit

Resuspending RNA in Formamide (as reported by Chomczynski et al. Nucleic Acids Research) has several benefits over storage in water or ethanol. First, formamide will protect RNA from RNases. The RNA is also extremely stable, allowing it to be stored overnight at 4C, or indefinitely at -20C. Samples can even be left at room temperature overnight without fear of degradation. Samples can be made to be highly concentrated, up to 4 mg/ml. Finally, with concentrated samples, no drying of your RNA is necessary for running Northerns, RT-PCR, or RNase Protection, saving time and potential degradation.

From CsCl, One Step Acid-Phenol, Trizol (tm) or RNazol B (tm) Extractions:

1. Precipitate your RNA as usual with either 100% ETOH (CsCl method) or Isopropanol (other methods). Spin to pellet.
   

2. Wash RNA pellet with 75% ETOH to remove excess salt...especially if using CsCl !!!
   

3. Aspirate ETOH, and make sure pellet is free of excess of ETOH, but DO NOT OVERDRY!!
   

4. Resuspend pellet in 100% formamide, which has been stored at 4C. Resuspend your pellet in anywhere from 10ul to 1000ul, depending upon pellet size.
   

5. Most or all of the pellet should dissolve instantly. Allow to sit at room temperature for 15 minutes. Pipet up and down frequently. If sample is very concentrated, allow to sit at 4C overnight.
   

6. To quantitate, OD 1ul in 500ul water. Remember to add 1ul of formamide to the blank as well to account for any background from the formamide.
   

7. Should the sample be too dilute, it can easily be precipitated like a water-solubilized sample, and resuspended in a smaller volume with 3M Sodium Acetate and 100% ETOH.
   

For QIAGEN RNeasy (tm) protocols:

1. After eluting the RNA from the columns with water, either dry down the sample, or precipitate with 3M Sodium Acetate and 100% ETOH.
   

2. Spin, resuspend in formamide, and OD

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Northern protocol for small RNA

10 N NaOH to pH 7.4

x mL

Type I water

1000 mL

Autoclave or filter sterilize. SSPE can replace SSC plus NaPO4 , pH 6.7.

50% Dextran sulfate MW 5000

Dextran sulfate MW 5000 works just as well as the traditional MW 500,000 material, but is significantly easier to dissolve. Weigh dextran sulfate directly into anti-static treated tube (do not try to transfer from weighing paper or weigh boat).

Dextran sulfate supports the growth of bacteria. Store at -20.

1. A Hoeffer/LKB Transphor unit is recommended for Northern blots. The Bio-Rad unit has been known to melt when incorrect voltage was applied.

The recommended transfer membrane for very small RNA was 0.1 nylon (Schleicher & Schuell Nytran or ICN Biotrans) but this material is no longer available. RNA that is < 110 nucleotides will tend to go through a 0.2 membrane. Try Schleicher & Schuell nitrocellulose membranes (BA79/0.1 � and BA75/0.05 )? Nylon (neutral or charged) membranes are rated for denatured > 50 bp DNA fragments, require alkaline transfer conditions and may have higher backgrounds.

Hoeffer sponges are 6?x 9?and the gel must be cut to fit these dimensions.

The Hoeffer gel box can blot 4 gels simultaneously if additional cassettes are purchased.

The Hoeffer unit generates more heat than the BioRad unit.

BioRad sponges are 6 1/8?x 8 3/8?and the gel must be cut to fit these dimensions. The BioRad box is limited to a single gel.

Polyacrylamide/TBE gels ranging from 4 to 8% have been successfully used, with or without urea. It is not necessary to presoak the gel to remove urea prior to blotting. If the gel is severely overloaded, some RNA will transfer to a second layer of nylon.

2. Water and buffers do not need to be DEPC-treated if they do not come from carboys. Carboy spouts tend to be a source of mildew RNases that are resistant to DEPC.

3. Prep a clean tray large enough to hold the blot cassette and soak in 0.1% DEPC or 3% hydrogen peroxide for 10 min at room temperature. Cover the pan during the soak.

4. Prep 5000 mL 1x TAE5 for the Hoeffer unit or 3300 mL 1x TAE5 for the BioRad unit. Pre-chill the buffer on ice in the coldroom.

5. Cut nylon or nitrocellulose and 2 pieces of Whatman 3MM to required dimensions while wearing gloves to prevent finger oils from coming into contact with membrane. Clip one corner to indicate orientation.

6. Drain 0.1% DEPC or 3% hydrogen peroxide from the tray. Rinse with a trace of 1x TAE5 . Add remaining portion of 1 liter 1x TAE5 to tray. Add plastic gel cassette and sponges. Squeeze the air out of the sponges. Soak the membrane briefly by laying it on the sponges.

7. Open the gel plates. Apply membrane to gel and gently brush out air bubbles. Dip one piece of 3MM very briefly in 1x TAE5 and lay on top of membrane. Remove air bubbles. Lay glass plate on 3MM and flip gel over. Remove the other plate (this seems to be difficult) and prewet the remaining piece of 3MM. Lay this on top of the gel. Remove air bubbles. Move to plastic cassette and assemble holder.

8. Place the membrane side of the gel cassette towards the red electrode. Add prechilled 1x TAE5 buffer to cover. Use the 1x TAE5 buffer in the presoak tray as part of the required buffer.

9. Careful! Electrode plugs on the BioRad Transblot will corrode when wet. Keep the buffer below the electrode plugs.

10. Place a magnetic stir bar in the bottom of the box. Move box to 4 coldroom. Buffer must be stirred during blotting run. Set magnetic stirrer at speed 6.

11. Hoeffer/LKB Transphor 10 v/30 min then 40 v/2 hours or 20 v/overnight

BioRad Transblot 20 v overnight. Do not use the 40 volt protocol with the BioRad unit.

12. UV fix the wet membrane (leave it on the wet 3MM support) with 120,000 oules UV via the UV crosslinker. Alternatively, use a Sylvania germicidal UV lamp exactly 12 cm from the bulb for exactly 2 min. Caution: nitrocellulose is highly flammable. Air dry the membrane. Place a single membrane in a hybridization bag. Seal the bag. Cut one corner and add prehyb solution at 5 mL/100 cm2 membrane. Incubate in shaking waterbath for 3 hours at 42 Cut a corner of the bag and remove all prehyb. Add hybridization solution at 5 mL/100 cm2 membrane.

13. Add 1 x 107 cpm 32 P-labeled probe to 300 reserved hyb mix and boil for 3-5 min. Transfer the probe to ice and then add the probe to the hybridization bag within 20 min. Seal the bag. Rinse the bag in the sink to remove prehyb or hyb mix from the outside of the bag, as these will support the growth of bacteria in the waterbath. Incubate the hyb solution in a shaking waterbath overnight at 55. Alternatively, a rotisserie apparatus can be used instead of hybridization bags and shaking water baths.

13. Prehybridization mix (add in order listed at 5 mL/100 cm2 membrane)

5x Denhardt, 5x SSC, 50 mM NaPO4 , pH 6.7, 50% deionized formamide250 /mL sheared salmon sperm DNA, 1% dextran sulfate MW 5000 and 0.1% SDS

Prep the hyb mix and prehyb mix fresh each time. The hyb mix can sit at room temperature during the 3 hour prehyb step.

14. Hybridization mix (add in order listed at 5 mL/100 cm2 membrane)

14. Hybridization mix (add in order listed at 5 mL/100 cm2 membrane):

1x Denhardt, 5x SSC, 20 mM NaPO4 , pH 6.7, 50% deionized formamide, 100 /mL sheared salmon sperm DNA, 5% dextran sulfate MW 5000, and 0.1% SDS

15. Wash square Pyrex dish that will fit in shaking waterbath. Soak in 3% hydrogen peroxide or 0.1% DEPC for 10 min.

16. High Stringency Washes 1, 2 and 3

Wash 1 2x SSC: 80 mL 25x SSC, pH 7.0, 910 mL Type I water, 10 mL 10% SDS

Wash 2 1x SSC: 40 mL 25x SSC, pH 7.0, 940 mL Type I water, 10 mL 10% SDS

Wash 3 0.5x SSC: 20 mL 25x SSC, pH 7.0, 970 mL Type I water, 10 mL 10% SDS

17. Discard the probe and hybridization solution in 32 P liquid waste. Rinse the bag with approximately 30 mL wash 1 and discard the wash in 32 P liquid waste. Discard the 0.1% DEPC in the tray and add approximately 100 mL wash 1 to the dish. Transfer the membrane to the dish and cover with Saran. Shake gently at room temperature for 10 min. Discard wash in 32 P waste. Microwave the remaining wash for 1 min to prewarm slightly and add to the Pyrex dish containing the probed membrane. Incubate at 65 for 30 min. Washes may be performed in the hybridization bag, but may result in higher background. Discard wash 1 and add 1000 mL prewarmed wash 2 and incubate at 65 for 30 min. Discard wash 2 and add 1000 mL prewarmed wash 3 and incubate at 65 for 30 min.

18. Air dry the filter on 3MM paper and wrap with Saran. Expose the membrane as desired.

Do not let the membrane dry out if reprobing is required. Place the blot in a clear plastic sheet protector and seal it closed with tape for chemiluminescent exposure. For storage, seal the blot in a water-tight seal-a-meal pouch and store at ?0. Note that only SaranWrap is water-tight. Other plastic wraps are water-permeable and allow blots to dry out (and allow water from wet gels to contact autoradiography film, causing black spots).

Neutral membranes have an equal and uniform distribution of charges. Positively charged membranes have a preponderance of positive charges across the matrix. They will hold the same amount of nucleic acid. Positively charged membranes have stronger retention: the target remains bound longer. UV crosslinking forms a covalent bond between nucleic acids and the positive charges of the membrane. Fixation by baking creates a hydrophobic interaction between the nucleic acids and the membrane. If stripping and reprobing is required, it is better to UV crosslink to a positively charged membrane in order to preserve the integrity of the target during the stripping step.

Quality membranes do not have a correct side. The charges are equally distributed on both sides.

Nominally, 0.22 pore membranes have greater surface area than 0.45 membranes. They also require better blocking. Consistent pore size is critical. For Northern blots of small RNAs (~ 100 nucleotides), 0.1 membranes are recommended.

Particulates in unfiltered buffer, skin oils, nonspecific antibody binding, addition of too much probe, inadequate post-hybridization washing, improper wash temperature, and other factors can create background fog or spots on membranes.

Blocking can reduce background. Non-specific proteins block certain electrostatic interactions which may attract proteins randomly. Milk proteins, BSA, or commercial blocking agents are typically used.