Extractionof25NTRNA

實驗概要

This  method is use to extract short RNAs from plant tissue. Some of the  variables (e.g. centrifugation speeds&times, precipitation times and  volumes of ethanol) have not been optimised: the conditions given here  work but I’m sure some streamlining is possible. There are three main steps: extraction, size fractionation, and final clean-up.

實驗原理

We originally estimated the size of the PTGS specific RNAs to be 25 nucleotides. I1ve found recently that like those in Drosophila cell there are actually two main species. In most plant silencing examples we1ve  looked at the smaller of the two is much more abundant and initially I  thought that the upper band was simply the trailing end of the lower  one. With some new systems we've been looking at the upper band is much  more pronounced relative to the lower and so its presence is much  clearer.Using RNA markers instead of DNA oligos and with the extraction  protocol outlined below the upper band runs at 25nt and the lower at 23  nt. this is still two nucleotides larger than the Drosophila RNAs  (21-23). However, when I gel purify the "25" and "23" and re-run them,  they run faster: two nt faster in fact! I1ll continue to refer to these as 25nt RNAs below for convenience.

25nt  RNAs can be detected from total RNA preparations (i.e. without  enrichment) in some cases. It depends on how clean your (this depends of  course on the plant type and extraction method)is and the strength of  the PTGS you are studying (stronger PTGS means more 25nt RNA and  therefore higher signal:noise). The choice is yours! Enrichment for low  mol.wt. RNA from total RNA preparations improves the ease of handling  and allows proportionally more to be analysed per assay than using total  RNA. Also, too much high molecular weight RNA and DNA in the samples  tends to cause drag during the run. Most of the types of plant tissue I  have used contain carbohydrates which co-purify with RNA under most  conditions and so I almost always take steps to remove these. The DEAE  step (step3) is also quite effective at removing polyphenols that  co-purify from some plant tissue.

主要試劑

SDS based extraction solution [ Details：see Science paper]

實驗步驟

1.  Start by extracting RNA as you would for total RNA: I use the SDS based  extraction solution (see Science paper) but others work too (e.g. GITC  (guanidine isothiocyanate), Kirby’s) and may even be superior. I do two  phenol/chloroform extractions of the slurry followed by precipitation  with ethanol (3 vol) and NaOAc (0.1 vol) for at least two hours at -20.  This is the basic total prep. Don’t use silica based columns (e.g.  "RNAeasy") because the short stuff doesn’t stick. Recover the total  RNA/DNA by centrifugation as you would normally (e.g. 10000xg/20 min).  If the ppt looks clean enough you could try going straight to the  Northern (after UV quant. of course).

2.  This next step is to remove the majority of the high molecular weight  nucleic acid. I’ve used several methods for this but this one is the  simplest and cheapest and may provide a low mol. wt. fraction that is  clean enough to use directly. I stopped using centricon filtration  because I found that their performance seemed compromised with extracts  from Nicotiana species. Another way I tried briefly was to use GITC  extraction solution, spin down debris, add 1/2 vol ethanol to s/n and  apply to a silica column (Supelco). This column bound all the RNA and  DNA bigger than about 200nt. I Phenol extracted the flow through, EtoH  pptd, and purified the lowMW RNA in the redissolved pellet away from the  contaminating carbohydrate by DEAE chromatography as below. This way  was a bit wasteful because of the large amounts of GITC solution and  phenol/chloroform I had to use so I did not pursue it.

My  current favourite method is as follows. Dissolve the pellet in water/TE  (volume depends on size of pellet; usually I use between 0.5 to 5 ml). I  heat this to 65 C to disrupt any association of the 25nt RNA with the  larger RNA and DNA molecules and it also quickens the dissolving of the  pellet. Place on ice and add PEG (MW=8000) to a final conc of 5% (I used  to use 10% but 5% is safer: less chance of small stuff co=precipitating  with large stuff) and NaCl to a final conc of 0.5M. Mix well, and leave  on ice for 30 mins. Spin down at 10000xg for 10 mins which will pellet  the high molecular weight nucleic acid. KEEP THE SUPERNATANT: this  contains mainly tRNA and small rRNAs but also the 25nt RNA. I also  normally dissolve the pellet of high mol.wt RNA/DNA in formamide to  compare with the subsequent low mol. wt. fraction in order to asses the  separation. Can also use this for conventional Northerns etc..

Add  three volumes of ethanol to the supernatant of the PEG pptn and place  at -20 for at least two hours. Spin down at 10000xg for 10 mins. This  ppt. comprises mainly tRNA and small rRNA and will run as a thick band  around 100-200bp on a non-denaturing mini-gel (e.g. one routinely used  for plasmid analysis). The high mol.wt. fraction (if you run it !) will  of course smear upwards from this. As I said, if the LMW fraction is  clean enough, I dissolve this in formamide and use it directly for  Northern blots. I quantify the low mol. wt. fraction either by taking  aliquots and measuring UV absorbance (after ethanol pptn to remove the  formamide) or by quantifying the fluorescence after EtBr staining of the  minigel. With Nicotiana species (for example from N. Benthamiana), I  get a lot of jelly like material (I thought this was likely to be  pectins but was assured by a cell wall expert that that was very  unlikely, so I don’t know what it is but its not RNA!) which causes  difficulty in handling and the RNA does not run well on the gel and so I  purify further as follows.

3.  I've recently started to use DEAE-sepharose CL-6b (Sigma) in preference  to Qiagen columns since it is cheaper. Equilibrate the sepharose in  buffer A (50mM MOPS/NaOH pH7; 15% isopropanol; 0.2M NaCl; 0.15% Triton  x-100) I make quick columns with this by cutting some whatman 1 filter  paper circles small enough to plug the end of a 5ml disposable syringe,  adding 1ml of the equilibrated sepharose and letting the column settle.  Redissolve the ethanol ppt’d low mol. wt. fraction in buffer A and apply  to column under force of gravity only. Or..you can skip a step by  adding water to the supernatant of the PEG pptn (section 2 above) until  the NaCl concentration reaches 0.2M and then adding 5-10 volumes of  bufferA and applying this to the column. Collect the flow-through and  reapply to column. Wash the column with more bufferA (I do 10 column  volumes) and elute with buffer B (same as bufferA except NaCl  concentration is 1.0 M). Add three vol of ethanol directly to this  eluate and place at -20 for at least two hours and after a 10000xg spin  for 20 mins and a 70% ethanol wash, I dry the pellets and redissolve  them in formamide (Usually 100-1000ul).

NORTHERN BLOTTING FOR LMW RNA

Gel:  15% polyacrylamide (19:1); 7M urea; 0.5xTBE. I use Biorad "protean"  apparatus = a vertical 500pxx500px glass plate gel sandwich with 1.5mm  spacers and a "castellation" type of comb.

Since  the samples are already in formamide, I heat these to 65 C for 5 mins,  place on ice, add 1/3 volume of 4xloading solution (= 2xTBE, 40%  sucrose, 0.1% bromophenol blue). I use anywhere between a few hundred to  50ug of RNA per lane.

Run  the gel in 0.5 x TBE at 100-500V until BPB is almost at the bottom  (usually 2-6 hours). Separate sandwich and place gel on a prewetted  (0.5xTBE) piece of HybondNX hyb1n membrane. I use a BIoRad "wet"  electroblotter to blot the RNA. I give it about 45 minutes at about 100V  in 0.5 xTBE. I used to use 33P kinased DNA oligos or DNA homologous to  your probe digested with a frequent cutter to give size markers. I now  use 32P labelled in vitro transcribed RNA which has been fully RNAaseT1  digested. After the blot, I place the membrane on several layers of  filter paper soaked in 20 xSSC to equilibrate the membrane. I don1t let  the solution of 20xSSC flood over the membrane surface because I1 m  worried that at this stage, prior to fixing, the attachment of such  small molecules to the membrane may not be particularly strong (this  could be a bit paranoid). After about 30 minutes, dry the membrane  thoroughly (e.g. in oven if you want). I then fix in a Stratalinker  (autocrosslink setting). Store membrane in dark until use.

Prehyb membrane in

50%  formamide; 7% SDS; 50mM NaHPO4/NaH2PO4 (pH7); 0.3M NaCl; 5x Denhardt1s  sol1n; 100ugml-1 sheared, denatured salmon sperm DNA, at 40 for at least  half an hour.

Probes:  I use single stranded, 32P labelled RNA probes transcribed in vitro  from plasmid templates. Initially, you should use the whole length of  the sequence undergoing PTGS since the 25nt RNA may not be derived from  the entire sequence. E.g.: In GUS PTGS, most of the signal comes from  the 31end of the GUS gene. Make the probe as you would for a normal  Northern. The specific activity is up to you but remember if you use too  little of your limiting NTP you may have underrepresentation of you  probe at its 31 end especially with longer probes. I remove the DNA  template by adding RNAase-free DNAase directly to the transcription  reaction. and continuing the incubation at 37 for an hour. Then I  hydrolyse the probe. This is very easy and the benefits are that I get  less background and also the membranes strip very easily. Make up a  200mM solution of carbonate buffer as follows: 80mM NaHCO3 (0.672 g per  100 ml)   120mM Na2CO3 (1.277 g per 100 ml). If you have done the  transcription in 20ul, add 300ul of carbonate buffer and incubate at 60  for as long as it takes to reduce the probe to an average size of 50-100  nucleotides. Use this formula to calculate the time

t= (Li - Lf)/(k.Li.Lf)

where

t = time in minutes

Li = initial length of probe in kb

Lf = final length of probe in kb (i.e 0.05 in our case)

k = rate constant = 0.11 kb. min-1.

It  usually takes 1-3 hours for the hydrolysis. Add 20 ul of 3M NaOAc (pH  5) to the reaction and then add all of this to the hyb solution. Hyb at  40 degrees C overnight, wash at 50 in 2xSSC/0.2% SDS. The strength of  signal of course depends on how much you load etc.. but I normally get a  clear picture after an overnight exposure with screens at -70. Patchy  background can arise from using too much probe: if you saved your probe,  simply strip the membrane (1 minute at 90 C in 0.2%SDS, 10mM Tris/HCl  pH7.5 usually completely strips these filters including background),  re-pre-hyb, add some of the hyb solution containing probe back and leave  to hyb again.

注意事項

There  appears to be some confusion arising from the way we have described the  protocol in the Science and Plant Cell papers.I hope the following  clears up any problems you have.

My  "Science" paper mentions using using ion exchange with Qiagen columns  to clean RNA preps up. I was referring to the original Qiagen support  which is a modified DEAE and a bona fide ion exchanger. I now use DEAE  sepharose which is cheaper and quite fine for separating small nucleic  acids. The modified DEAE of Qiagen was, I think, the product upon which  the Qiagen company was founded (the modification they made made it  possible to use DEAE for separating much larger nucleic acids such as  plasmids hence the success of the company). More recently Qiagen (along  with some other companies) have introduced silica based RNA purification  kits. This idea is just an extension of original observations years ago  that DNA binds silica (the basis of "Geneclean" gel purification kits).  These were never much use for RNA until it was noticed that you could  get RNA to bind to silica by adding ethanol to the mixure of silica,  chaotropic salt and RNA. I don't know who owns the IP on this but I  don't think Qiagen invented it. At any rate they now produce these  silica based purification kits which are fine for mRNAs. However, as we  say in Tamas' "Plant Cell" paper, the short RNAs do not stick to these.  They are not really ion exchange columns so it should have been possible  with very careful reading of the two papers to work it out but I do see  how the confusion has arisen.