My experience (Trustworthy protocols for biology)

Crosslinked RNA Immunoprecipitation

2009-08-27T16:19:00.007+02:00

preliminary comment:

The following text is taken from my Materials and Methods, so please excuse the raw way of writing.

Aim of the technique:

This technic allows for the visualization of how well a known RNA interacts with a know protein in the presence of its partners (the partners of the RNA binding protein of course).

Brief introduction:

- You incubate a radioactive RNA with a protein extract

- specifically crosslink the RNA-protein interaction with UV light (254 nm)

- digest unbound parts of the RNA so that only a few radioactive and non-radioactive nucleotides remain bound to the protein (little variation in the size of the protein makes it easier to be identified while running the samples on a gel). Alternately, you can confirm the protein's identity by immunoblotting.

-Immunoprecipitate your protein of interest, crosslinked to the radioactive nucleotides and identify it by running samples on a SDS-PAGE and autoradiography.

Enough talking, here is the protocol:

required products:

-nuclease-free water (prepare DEPC-treated water: incubate 1L of water with 0.1% v/v of diethyl pyrocarbonate overnight at 37°C with vigorous stirring and autoclave it)

-Dynabeads protein A (also test protein G coupled beads which may work better depending on the class of antibody you want to immobilize)

- PBS,

- molecular grade BSA powder

- Tris-HCl pH 7.9,

- NaCl,

- Nonidet P40

- radioactive body-labeled RNA (obtained by in vitro transcription with alpha P32 UTP)

Avoid 5'end labeled RNA if your not sure that your protein binds on the first nucleotide of your RNA)

tips before starting:

-don't forget to load the input lane corresponding to each immunoprecipitation you present (composed of the radioactive RNA+ protein extract treated with RNase A)

-don't forget to do a negative control (isotype control antibody: same class as your antibody, but not raised against any antigen). A negative control without antibody bound to the beads won't work here due to physical properties of the Dynabeads, which is also the reason why I tried to decrease non-specific binding of RNA and proteins to the beads by pre-blocking them TWICE! with BSA.

-It goes without saying that all solutions are better working when prepared freshly and that all solutions need to be prepared in nuclease-free water.

protocol:

200,000 cpms of a radioactively body-labeled RNA were washed in 70% ethanol, dried and resuspended in 16 µL water. To this solution, 10 µg of yeast tRNAs and 20 µL of a nuclear extract prepared from HeLa cells (~30 µg/µL)were added. This mixture was incubated for 15 min on ice, exposed to UV light for 15 min on ice (UV Stratalinker™ 2400, lambda=254 nm, Stratagene), and treated with Rnase A at a 1 µg/µL final concentration for 15 min at room temperature. Meanwhile, 20 µL of a 50% suspension of Dynabeads coupled to protein A (Invitrogen) were washed once with 1 mL PBS and pre-blocked 30 min at room temperature with 0.2% BSA complemented PBS buffer under gentle rotation (16 rpms). The resin was washed three times with 1 mL of NET-2 buffer (50mM Tris.HCl pH 7.9, 100 mM NaCl, 0.05% NP40, 0.5 mM DTT) and pre-blocked again for 1 hr at 4°C in 5% BSA complemented PBS. Following three successive washes in NET-2 buffer, the beads were resuspended in a final volume of 42.5 µL of NET-2 buffer and incubated for 1 hr at room temperature with 3 µg of antibody, under gentle rotation. Antibody-coated beads were washed three times in 1 mL of NET-2 buffer and incubated for 1 hr at 4°C with to the former mixture of nuclear extract with radioactive RNA and tRNA, under gentle rotation. The unbound proteins were washed from the beads three times with 1 mL of NET2 buffer and elution occurred by addition of 12 µL of 1X sample solution and heating 5 min at 95°C.

Don't hesitate to comment or contact me for troubleshooting.

RNA affinity chromatography

2009-07-10T15:12:00.016+02:00

This is a protocol for RNA immobilization of two in vitro synthesized RNAs (RNA A and RNA D) of respectively 61 and 73 nt on agarose beads and purification of RNA-bound proteins, according to Caputi et al. 1999

It was done with 5'end labeled RNA to test the efficiency of RNA immobilization on the agarose and the cpm counts are displayed on the right side of the excel worksheets.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=10406810

P.S.: I implemented NP40 in the washing buffer of the last downloadable protocol, allowing for more stringency than what is illustrated by these two gels.

purification of in vitro transcribed RNA

2009-05-21T16:03:00.011+02:00

For this you will need to run you transcription product on a denaturing acrylamide gel:

N.B.: Urea takes time to dissolve and may require gentle warming (40-50°C) so try to prepare the two urea containing solutions in advance.

solutions to prepare

10% w/v ammonium persulfate in water (can be stored at 4°C)

100 mL 20% acrylamide/urea solution:

49.85 g UREA

0.5 g N,N methylene bisacrylamide

50 mL acrylamide 40% stock (Bio-Rad 161-0140)

10 mL TBE 10X

TBE 10X :

108 g Tris base

56 g boric acid

40 mL 0.5 M EDTA pH8.0

double distilled H2O (ddH2O) to 1L

100 mL of TBE/UREA solution:

dissolve 49.85 g UREA in 80 mL autoclaved H2O

add 10 mL 10X TBE

top up to 100 mL with autoclaved H2O

formamide loading buffer:

10 mL formamide

10 mg xylene cyanol FF

10 mg bromophenol blue

200 µL 0.5 M EDTA pH8.0

Casting and pouring the gel

Choose the adapted % for your transcript :

dye migration according to denaturing acrylamide %

Then cast a 1 mm x 20 cm x 20 cm gel, taking care to clean both glass plates an

d silanizing one of the two plates (silanizing liquid available from Lonza).

gel recipe:

X mL acrylamide/UREA8.3M

TBE/Urea to 40 mL final

240 µL 10% ammonium persulfate

50 µL TEMED

This gel polymerizes very fast due to the presence of urea, be careful !!!!!

Avoid leakage by pouring the gel as horizontally as possible, bubble formation should be avoided but if bubbles appear, you can always try to get rid of them by playing with the left/right inclination of the assembled glass mold.

clean the wells with a syringe equipped with a bent needle.

pre-run the gel in TBE 1 X for 30-60 min at 500V

Sample preparation

During the pre-run, add 40 µL of loading buffer to the 50 µL transcription product redissolved in EDTA and heat 5 minutes at 95°C,

chilled on ice,

stop the pre-run f the gel,

clean the wells a second time, with a syringe equipped with a bent needle prior to load,

load the samples carefully and run the gel keeping the same conditions as in the pre-run.

Monitor RNA

RNA should be accurately visualized by UV shadowing : using a UV lamp (I used 254 nm to better see the RNA, but higher lambdas are considered less damaging for RNA) and a silica coated plastic sheet (usually used in Thin layer chromatography).

Uncast the gel when the run is over and dispose it on a cellophane film, put the silica sheet under this cellophane and directly expose the gel to the UV source, which gives this from top to bottom:

UV light

gel

cellophane

silica

Alternately, if the RNA was transcribed with radioactive alpha 32P UTP, you just have to monitor its presence by autoradiography.

Cut the band of interest, chop it in very small slices.

Elute it in elution buffer:

elution buffer can vary depending on the type of experiment that follows the purification.

If you want to 5'end label the RNA for instance it is advised not to use tRNA in the elution buffer, whereas this is not a problem for RNA that has been body labelled with radioactive UTP during transcription.

For 5' end labeling it is also important NOT TO USE DEPC anywhere since this compound may inhibit dephosphorylation and phosphorylation steps.

elution buffer with tRNA (400 µL should be sufficient) :

0.75M ammonium acetate

0.1% SDS

10 mM MgOAcetate

0.1 mM EDTA pH8.0

add at the last minute

25 ng/µL bovine or E.coli tRNA

10 µL/mL phenol buffered with 0.1M citrate pH 4.3

elution buffer without tRNA (1 mL should be sufficient):

50 mM Tris-HCl pH7.5

300 mM NaOAcetate

0.5% SDS

Incubation can vary but minimum is of 2 hrs at room temperature (tried with no tRNA) or 37°C (tried with elution buffer with tRNA and phenol) under vigorous shaking.

Once elution is performed, treat with acidic phenol, chloroform and precipitate overnight with NaOAc and pre-chilled absolute ethanol.

Avoid using ammonium acetate if the RNA is prepared to be used for radioactive 5' end labeling.

resuspend RNA in autoclaved water.

large scale T7 in vitro transcription (1 mL)

2009-05-21T15:24:00.008+02:00

1°) Use autoclaved water that can in addition be treated with Diethyl pirocarbonate to remove pyrogens and potential RNases:

Add 0.1% DEPC (vol./vol.) in the water and incubate overnight at 37°C, with a rotating magnetic stirrer. Autoclave water . (DEPC, Sigma D 5758)

2°) Prepare 10X buffer: 400 mM Tris-HCl pH8.1

10 mM spermidine

3°) Assemble the reaction described below respecting the same order:

422 µL H2O

100 µL 10X buffer

80 µL ATP 50 mM

80 µL CTP 50 mM

80 µL GTP 50 mM

80 µL UTP 50 mM

28 µL MgOAcetate 1M

100 µL DTT 50 mM

10 µL Triton X100 1%

10 µL DNA template* 5 µM

10 µL T7 RNA pol. (home made but follow manufacturer instructions for commercial enzyme)

*: the type of template can vary here: linearized vector, annealed oligos

4°) Incubate 5 hrs at 37°C

if reaction works, the reaction mixture should turn with turbid due to accumulation of PPi, can start 30-60 minutes after reaction started.

5°) Treat with DNase 1 (determine the number of units needed according to the amount of template used in the reaction and to the manufacturer's suggestion). The enzyme should work in the transcription buffer.

6°) Denature proteins, volume to volume, with phenol buffered with 0.1M citrate pH4.3 (Sigma P 4682) (i.e. add 1mL to the transcription reaction).

7°) Extract with chloroform, volume to volume.

8°) Transfer aqueous phase to a high centrifugation speed tube (i.e. COREX if available) and precipitate overnight

with parafilm to close the tube:

1 mL Transcription reaction

111 µL NaOAcetate 3M pH5.2

3.350 mL absolute EtOH (precooled at -20°C)

9°) centrifuge 30 minutes , 10 000xG at 4°C

10°) the pellet obtained should contain both the RNA and the PPi that will make it look solid, redissolve it in 0.5M EDTA pH8.0 , 50 µL should be sufficient here. Don't shake the redissolving pellet on a vortex, but prefer to leave it dissolving at room temperature for a while without shaking. Handshake the tube to control dissolution. Partial dissolution of the pellet is not a problem since RNA should redissolve in water anyway.

acknoledgements: Camille Mary

Explanations

2009-05-02T19:04:00.003+02:00

Hi all,

In this blog I will (try to) post protocols that I ve used or still use, and were set up by me or reliable friends.

Your protocols are also welcome as long as they can be proven to actually work ! (no defined clear way to do so yet, but I will think of it).

All advices are welcome as long as politely mentioned and you can contact me for any detail regarding protocols such as brands and concentrations (unit/microL etc..).

But remember that if results are not positive does not necessarily mean that the experiment didn't work.

Wishing you all to have successful experiments.

Nicolas.

PCR tips and troubleshooting

2009-05-02T10:35:00.008+02:00

I will start this blog with PCR, not giving a protocol this time but guidelines and tips that worked for me or colleagues.

PCR seems easy at first sight, but it can turn out to be nightmarish for obscure reasons.

As a first try I will thus try to summarize my own experience of PCR.

PCR reaction usually consists in 5 steps:

a first long denaturation step at 94°C (some do it at 95 or even up to 98)

then iterative cycles of temperatures:

94/95 for 1 minute or less

50 for 1 minute or less

72/68 for n minutes (depending on size of amplicon)

and finally a step of 10 minutes at 72°C

The first step doesn't need to be too long 2 minutes should be much more than enough.
Then, the cycles can be repeated as much as 35 to 40 times, after that the "thermoresistant" polymerase should be damaged due to repeated denaturation steps, which is also why this step should not be too long (30 sec to 1 min).

The transition between denaturation and annealing is VERY important, despite what some could think, the slower this step occurs the more specific should be the annealing. However, it is to be noted that this transition allows for a short period of time spent at the polymerization temperature of the polymerase e.g. 72/68°C so it cannot be too long either. For tricky PCRs I went as low as 1°C/sec decrease speed, instead of 3°C/sec normally used.

The annealing step:

The annealing temperature is also important thought it should allow for the template-complementary part of your primers to anneal to the template. For this step one should check that the forward and reverse primers are thermodynamically OK (check for concatemerization and other stuff on Netprimer, link on right hand of this page in "useful links"). Design of primers can be done through online Primer 3 service.

If the energy of a false priming or self annealing is 4 to 5 times less than the proper annealing energy then it's fine. Unfortunately I don't keep track of how bad were the ratios of my PCRs, but I will give tips on improving desperate PCRs.

Then the annealing temperature should be determined by calculation of melting temperatures of both primer, take average and remove 2°C. If they are too far from each other, just take the lowest one and remove 2°C.

The polymerization step:

This step is the least difficult to set up, just look at the datasheet provided with the polymerase of your choice and it should tell you how long and at which temperature polymerization is best achieved.

The last step is the 72°C 10 minutes. This step is in my opinion a folkloric step, that I already tried removing with success, but for the sake of traditions and also since I did it for all previous PCRs and I usually repeat them to compare results, I keep on adding it. But if you think of it this step is ridiculous since primers would not anneal properly to badly polymerized ends, so it makes no sense, in my opinion again, to add this long step at the end.

Optimizing your PCR:

1°) do it with different annealing temperatures and pick the best. If you don't have time for that or you don't need this PCR for precise amplification but rather to obtain a DNA for cloning, then you should try touch-down PCR (start your cycles with an annealing temp. higher than calculated and incremently decrease it at each cycle so that stringency of annealing progressively decreasses, allowing for only proper annealing in the first place so that amount of good template is largely increased when conditions are less stringent and it is then the major product of amplification).

2°) Once annealing temperature is optimized you can step up to optimizing MgCl2. Magnesium is a divalent cation that will allow negative charges of the DNA phosphate backbone to face each other when annealing. The lower this concentration is, the more specific should be the amplification of the products.

3°) You should also try to have the lowest amount of primer possible, so that primer don't dimerize due to excess, this can easily be monitored by looking at the bottom of your gel : round shaped halo, or big potato sometimes. 10 pmoles of each primer per PCR should be sufficient and I actually go for 5 pmoles in some of mine.

4°) If you need your PCR product for cloning and encounter troubles during further ligation steps, consider switching from TBE to TAE for running and buffering your agarose gels, borate is known to inhibit ligase.

5°) Think that the enzyme is provided in glycerol and it needs to be properly diluted to proceed through asmplification, too much glycerol may cause problems so take good care to dilute your reaction as advised by manufacturer.

6°) Too much DNA template may also prevent amplification, try testing series of dilutions of your template in the same PCR conditions.

7°) don't optimize too many things at a time, unless they were all independently shown to improve your PCR.

If after this you still get non specific things, than you can try hot start (adding the enzyme only at the 72°C step of the first cycle). You should also make sure that no reagent is contaminated (i.e. do a negative control without template) and if no polymerization occurs try a positive control of touch-down PCR.

When these things are checked and it still doesn't work, consider testing other polymerases and optimizing them. When things were desperate (I mean 6 or 8kb of genomic DNA to be amplified with no mistake allowed) I've used an enzyme from TaKaRa called HS LA Taq (HS: hot start, LA : long accuracy) *I'm not from TaKaRa Corp. and I usually don't like to advertise, but this brand is really worth it because not only do they sell briliantly working enzymes, but you can get sample quantities of it so you don't get charged like hell*. Some enzymes are sold with an antibody blocking them so that they only start amplifying once the antibody is destroyed after denaturation step, they are called hot start and to my experience they work better than any "melting wax" (used in the old times to prevent contact of polymerase with nucleic material before denaturation ocured) or "addition of enzyme at 72°C".

It is also possible to lower annealing temperature when too high with chemicals such as formamide. Decrease of annealing temp. due to formamide presence is best described by the following equation:

TFm = Tm - 0.61(%formamide, w/v)

Others have tried different compounds (Chakrabarti and Schutt 2001 NAR , for example)

Consider also that your template may be GC rich. I never faced that problem myself so I cannot help much with that, but polymerases are sold for these particular situations.

(see Hubé F, Reverdiau P, Iochmann S, Gruel Y. Mol Biotechnol. 2005)

Don't hesitate to post any questions or ask for advice.

Good luck !

I hope this helps