5¢end cDNA amplification using classic RACE
Elizabeth Scotto–Lavino 1,2,Guangwei Du 2&Michael A Frohman 1–3
Graduate
Program in Molecular &Cellular Pharmacology;Department of Pharmacological Sciences &Center for Developmental Genetics,Stony Brook University,
Stony Brook,New York 11794,U.S.A.3Correspondence should be addressed to M.A.F.(michael@pharm.stonybrook.edu).Published online 29December 2006;doi:10.1038/nprot.2006.480
The 5¢ends of transcripts provide important information about transcription initiation sites and the approximate locations of local cis -acting enhancer elements;it is therefore important to establish the 5¢ends with some precision.RACE (rapid amplification of cDNA ends)PCR is useful for quickly obtaining full length cDNAs for mRNAs for which only part of the sequence is known and to identify alternative 5¢or 3¢ends of fully sequenced genes.The method consists of using PCR to amplify,from complex mixtures of cellular mRNA,the regions between the known parts of the sequence and non-specific tags appended to the ends of the cDNA.
Whereas the poly(A)tail serves to provide such a tag at the 3¢end of the mRNA,an artificial one needs to be generated at the 5¢end,and various approaches have been described to address this step.The classical scheme for 5¢RACE described here is simple,suffices in many instances in which RACE is needed and can be performed in 1–3days.
INTRODUCTION
Most attempts to identify and isolate a novel cDNA result in the acquisition of clones that represent only a part of the mRNA ’s complete sequence.The ever-growing collections of sequenced genomes and high-quality cDNA libraries can often facilitate acquisition of the remainder of the transcript.For less well-characterized organisms,or for low-abundance cDNAs even in well-characterized organisms,such information is often not avail-able,particularly at the 5¢end of the transcript.Obtaining a full-length cDNA at the 5¢ensures that the entire protein region has been identified and yields information concerning the transcription initiation site.In some instances,5¢untranslated regions encode structural information that is relevant to mRNA stability,restricted subcellular localization or translational efficiency.
The missing sequence (cDNA ends)can be cloned by PCR,using a technique variously called rapid a
mplification of cDNA ends (RACE)1,anchored PCR 2or one-sided PCR 3(Fig.1).Since the initial reports describing this technique,many labs and com-panies have developed significant improvements on the basic approach 4–15.A protocol for 5¢end cDNA amplification by classic RACE is presented here.3¢end cDNA amplification can also be performed using a classic RACE protocol as described in a separate protocol 16.A more complex but also more powerful approach (new RACE),which has evolved from the work of a number of laboratories 17–24is also described in a separate proto-col 25.Commercial RACE kits and libraries are available from many companies that are more convenient but often not as powerful as the versions described here.
Classic RACE
PCR is used to amplify partial cDNAs that represent the region between a single point in an mRNA transcript and its 3¢or 5¢end
(Figs.1,2).A short internal stretch of sequence must already be known from the mRNA of interest.From this sequence,gene-specific primers (GSPs)are chosen that are oriented in the direction of the missing sequence.Extension of the partial cDNAs from the unknown end of the message back to the known region is achieved using primers that anneal to the pre-existing poly(A)t
ail (3¢end)or an appended homopolymer tail or linker (5¢end).Using RACE,enrichments in the order of 106–107-fold can be obtained.As a result,relatively pure cDNA ‘ends’are generated that can be easily cloned or rapidly characterized using conventional techniques 1.T o generate ‘3¢end’partial cDNA clones,mRNA is reverse transcribed using a ‘hybrid’primer (Q total ;Q T )that consists of two mixed bases (GATC or GAC followed by (T)17followed by a unique 35-base oligonucleotide sequence (Q I –Q O ;Fig.2a ,c ).Amplification is then performed using a primer containing part of this sequence (Q outer ,Q O ),which now binds to each cDNA at its 3¢end,and using a primer derived from the gene of interest (GSP1).A second set of amplification cycles is then carried out using ‘nested’primers (Q inner (Q I )and GSP2)to quench the amplifica-tion of non-specific products.
T o generate ‘5¢end’partial cDNA clones,reverse transcription (primer extension)is carried out using a gene-specific primer (GSP-RT;Fig.2b )to generate first-strand products.Following this,a poly(A)tail is appended using terminal deoxynucleotidyl-transferase (Tdt)and dATP .Amplification is then achieved using the hybrid primer Q T to form the second strand of cDNA,the Q O primer,and a GSP upstream of the one used for reverse transcrip-tion.Finally,a second set of PCR cycles is carried out using nested primers (Q I and GSP2)to increase the yield of specific product 4.Updated RACE Techniques
The most technically challenging step in classic 5¢RACE is to cajole reverse transcriptase to copy the mRNA of interest in its entirety into first-strand cDNA.Because prematurely terminated first-strand cDNAs are tailed by terminal transferase just as effectively as full-length cDNAs,cDNA populations that are composed largely of prematurely terminated first strands will result primarily in the amplification and recovery of cDNA ends that are not full length (Fig.3a ).This problem is regularly encountered for vertebrate
p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s
/m o c .e r u t a n .w w w //:p t t h mRNA
Partial cDNA clone
5′ UTR
Coding
3′ UTR
Figure 1|A schematic representation of the setting in which Classic RACE is used.The figure shows a mRNA for which only a partial,internal cDNA is available.
NATURE PROTOCOLS |VOL.1NO.6|2006|2555
PROTOCOL
genes,which are often GC-rich at their 5¢ends and,therefore,often contain sequences that hinder reverse transcription.A number of laboratories and companies have developed steps or protocols that are designed to overcome the problem 17–24.
New RACE.One approach to force the specific acquisition of full-length 5¢cDNAs consists of ligating an anchor primer to the 5¢end of the mRNA before performing the reverse transcription step (Fig.3b )17.Accordingly,subsequently generated cDNAs that do not extend all the way to the 5¢end of the transcript fail to incorporate the anchor sequence and do not get amplified in the ensuing PCR mediated by the gene-specific and anchor primers.This method,which is discussed in an accompanying protocol 25,is more powerful than the classic 5¢RACE protocol described here,but is also more challenging to perform.
Cap-switching RACE.A simpler method to amplify only full length cDNA ends involves adapter addition during reverse tran-
scription (cap-switching RACE;Fig.4).This method takes advantage of the propensity of Moloney murine leukemia virus reverse transcriptase to add an extra 2–4cytosines to the 3¢ends of newly synthesized cDNA strands upon reaching the cap structure at the 5¢end of the mRNA template 26,27.In the presence of a primer terminating in multiple Gs at its 3¢end,annealing and then complementary copying of the sequence of the annealed oligo takes place,which adds a linker sequence to the cDNA terminus.Because the template-independent addition of cytosines is cap-dependent,the oligo is appended only to full-length cDNA ends.Also,because this method involves fewer steps than classic and new RACE,it is simpler;however,the presence of the dG-terminating (‘switch’)primer can cause problems if it binds to C-rich sequences in the mRNA of interest.
Making cDNA ends meet.Another variation of RACE allows for the simultaneous amplification of both ends of a cDNA molecule,eliminating the need for performing two separate 5¢and 3¢RACE reactions 21,28.One version of this approach 28is achieved through a combination of a standard reverse transcription template switching (TS)reaction —in which a so-called TS-oligo is added that allows the reverse transcriptase enzyme to switch templates from the mRNA to the oligonucleotide,creating a double-stranded molecule —and inverse PCR,with a crucial ligation step between them.The ligation reaction circularizes the double-stranded cDNA,allowing primers that are directed away from the unk
nown sequence to be used.This straightforward method has been reported to compare very favorably to standard RACE techniques with respect to sensitivity and specificity 28.
自控Commercially available RACE kits and their limitations.Var-ious commercial RACE kits are available,including Clontech’s cap-finding (switching)Smart RACE system,Invitrogen’s 5¢RACE
p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s
/m o c .e r u t a n .w w w //:p t t h mRNA
Reverse transcription
1st strand cDNA
GSP1
GSP2
First set of amplifications
Second set of amplifications "cDNA 3′ End"
mRNA Reverse transcription
1st strand cDNA
GSP-RT GSP-RT
cDNA tailing 1st strand cDNA
GSP-RT First set of amplifications
Second set of amplifications
新
贸易保护主义GSP1
GSP2
"cDNA 5′ End"
Xho I Sst I Hind III
Q T
Q O
Q I
Q I
Q T Q O
周昌贡Q O-Q I-
Q T Q O Q T
Q O
Q O
Q I Q I
*
3′
5′
*
a
b
c
Q I
Figure 2|A schematic representation of Classic RACE.Please see text for details.(a )Amplification of 3¢partial cDNA ends.(b )Amplification of 5¢
partial cDNA ends.(c )Schematic representation of the primers used in Classic RACE.The 52nucleotide QT primer (5¢QO-QI-TTTT 3’)contains a 17-nucleotide oligo-(dT)sequence at the 3¢end followed by a 35-nucleotide sequence encoding Hind III,Sst I,and Xho I recognition sites.The QI and QO primers overlap by a single nucleotide;the QI primer contains all three of the
restriction enzyme recognition sites.Optionally,two additional nucleotides can be added to the 3¢end of QT to force it to bind to the junction of the cDNA and the poly(A)tail:(G,A or C,followed by G,A,T or C).Primers:QT:5¢-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3¢QO:
5¢-CCAGTGAGCAGAGTGACG-3¢QI:5¢-GAGGACTCGAGCTCAAGC-3¢GSP1,gene-specific primer 1;GSP2,gene-specific primer 2;GSP-RT,gene-specific primer,used for reverse transcription;*-,GSP-Hyb/Seq (a gene-specific primer for use in hybridization and sequencing reactions).
Classic RACE
New RACE mRNA
mRNA
Reverse transcription
GSP-RT
Ligation of RNA oligo
mRNA
Reverse transcription
GSP-RT
Poly(A)tailing
hdtune2.52
*
*****
a
b
Figure 3|The advantage of new RACE over classic RACE.(a )In classic RACE,premature termination in the reverse transcription step results in polyadenylation of less-than-full-length first-strand cDNAs,all of which can be amplified using PCR to generate less-than-full length cDNA 5¢ends.The asterisk indicates cDNA ends that will be amplified in the subsequent PCR.(b )In new RACE,less-tha
n-full-length cDNAs are also created,but only full-length molecules are terminated by the RNA oligonucleotide (the anchor sequence)and hence amplified in the subsequent PCR.
2556|VOL.1NO.6|2006|NATURE PROTOCOLS
PROTOCOL
system,and Ambion’s First-Choice RLM-RACE kit.The Ambion kit has been a popular choice for validating micro RNA (miRNA)cleavage sites 29,30.Commercial systems are often geared toward the construction of universal pools of full-length cDNAs (Fig.4),in which all of the mRNAs in the starting material become converted to cDNA.The value of this approach is that a single reverse-transcription pool can,in theory,be used to obtain the 5¢end of any transcript.By contrast,non-commercial versions of RACE have emphasized the use of a GSP to generate the first-strand cDNA templates.Although it lacks universality,the latter approach is more powerful because the reverse transcription step starts closer to the 5¢end of transcript,and the relative frequency of the desired cDNA is increased 450-fold in the resulting pool.This greatly increases the chances of the desired 5¢end being present in sufficient quantity to be amplified using standard PCR methods.Which approach should investigators choose?For the one-time user or for those with limited molecular biology experience,the most practic
al approach would be to obtain a commercial system and,if possible,a pre-made pool of reverse-transcribed cDNAs.Pools representing many human tissues are available —for example,from Clontech or Origene.Failing that,using a GSP-RT primer with the commercial kits will overcome the limitation described above.The Clontech and Ambion systems are relatively powerful and easy to use (both are variations on new RACE);however,they may not be optimal for every purpose.Invitrogen’s system is simpler and less powerful (a variation on classic RACE),but may suffice for many needs.In addition,because the commer-cial kits are relatively expensive,investigators who plan to use RACE regularly will achieve substantial savings if they prepare the reagents themselves.
Experimental design considerations for 5¢RACE
Reverse transcription reaction.In 5¢end cDNA amplification,the efficiency of cDNA extension is crucial.In the classic 5¢procedure,each specific cDNA,no matter how short,is tailed and becomes a potential target for amplification (Fig.2a ).Thus,the quality of the final PCR products directly reflects that of the reverse transcription reaction.The length of the first-strand cDNA can be maximized by using clean,intact RNA,and by selecting a reverse transcriptase primer that anneals near to the 5¢end of a region of known sequence.Improvements can also be made,at least in theory,by using a combination of SuperScript II and heat-stable reverse transcriptase at multiple temperatures.At incre
ased tem-peratures the amount of secondary structure encountered in GC-
rich regions of the mRNA should be reduced.Incorporation of cyclic compatible solutes such as homoectoine can also improve the generation of first-strand cDNA or the subsequent PCR amplifica-tion steps 31,32.
Poly(A)tailing reaction.T o attach a known sequence to the 5¢end of the first-strand cDNA,a homopolymeric tail is appended using Tdt.It is preferable to add poly(A)tails 4rather than poly(C)tails 2for a number of reasons.First,the 3¢end strategy is based on the naturally occurring poly(A)tail;adding a poly(A)tail to the 5¢end allows the same adapter primer to be used for both ends,which simplifies the protocol and reduces the cost.Second,because A:T binding is weaker than G:C binding,longer stretches of A residues (approximately two times longer)are required before the oli-go(dT)-tailed QT primer will bind to the template.Internal poly(A)tracts are rare so the chance of non-specific binding and the production of truncated amplification products is reduced.Third,vertebrate coding sequences and 5¢untranslated regions tend to be biased toward G/C residues;therefore,use of a poly(A)tail further decreases the likelihood of inappropriate amplification.Unlike many other applications that use homopolymeric tails,the actual length of the tail added here is unimportant,as long as it exceeds 17nucleotides.This is because the oligo(dT)-tailed p
rimer binds at the junction of the appended poly(A)tail and the cDNA transcript.The conditions described in the procedure result in the addition of 30-400A residues.
Many of the remarks made above apply also to the protocol on amplifying 3¢-end partial cDNAs 16and should be noted.There is,however,one major difference.The annealing temperature in the first step of 5¢RACE (481C)is lower than that used in successive cycles (52–681C).This is because cDNA synthesis during the first round depends on the interaction of the appended poly(A)tail and the oligo(dT)-tailed QT primer.In all subsequent rounds,ampli-fication can proceed using the QO primer,which is composed of B 60%GC and which can anneal to its complementary target at a
p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s
/m o c .e r u t a n .w w w //:p t t h Reverse transcription
Biotin
P i
P O -5′
P Totalibm as400
Biotin
P i P O -5′
Template switch
Biotin
3′ -RACE
GSP1
旧唐书李白传GSP2GS-Hyb
P O
P i
5′ -RACE RT
5′-U O
U i
3′
5′-U O U i
3′
Cap
5′-U O U i
3′
3′-U O N i Cap
U O
U i
GSP-Hyb
RGSP1
RGSP2
Cap
P i
P O -5′-a
b
c Figure 4|Schematic representation of cap-switching RACE.(a )Reverse transcription,template switch an
d incorporation of adaptor sequences at th
e 3’-end o
f first strand of cDNA.Biotin-labeled primer P total is used to initiate reverse transcription through hybridization of the poly(dT)tract with the mRNA poly(A)tail.After reachin
g the 5¢end of the mRNA,oligo(dC)is added by reverse transcriptase in a cap-dependent manner.Following this,throug
h template switch via base-pairing between the oligo(dC)and the oligo(dG)at the end of cap finder Adaptor,the reverse complementary sequence of the cap finder primer is incorporated into the first strand of the cDNA.Dotted line,mRNA;solid line,cDNA;rectangle,primer.The bracket indicates the known region.(b )The first round of PCR uses primer U o and RGSP1(reverse gene-specific primer 1),the 2nd round,U
i and RGSP2.GSP-Hyb is also within the known region,and it can be used to confirm the authenticity of the RACE product.(c )3¢-RACE.Reprinted with permission from ref.35.
NATURE PROTOCOLS |VOL.1NO.6|2006|2557
PROTOCOL
much higher temperature.In practice,the ideal melting tempera-tures will vary with individual PCR machines made by different companies.
Here we provide a detailed protocol for classic RACE,describing the reverse transcription steps,addit
ion of the poly(A)tail and the subsequent rounds of PCR amplification.
MATERIALS
REAGENTS
.dNTP solution (containing all four dNTPs,each at 10mM).DTT (0.1M)
.Tris–EDTA solution (10mM Tris-HCl [pH 7.5],1mM EDTA [pH 8.0]).Reverse transcription buffer,5x (as supplied by manufacturer).RNase H .RNasin
.SuperScript II reverse transcriptase (Invitrogen)
.Gene-specific primer,used for reverse transcription (GSP-RT primer;100ng/m l)
.Poly(A)+RNA,or total RNA.Poly(A)+RNA is used in preference to total RNA for reverse transcription to reduce background,but it is unnecessary to prepare it if only total RNA is available .CoCl 2(25mM)
.dATP solution (1mM)
.T erminal deoxynucleotidyltransferase (Tdt,Invitrogen or Boehringer Mannheim)
.Tailing buffer,5x (125mM Tris-HCl,pH 6.6,1M potassium cacodylate,1250m g/ml BSA)
.Hercules Hot-Start polymerase buffer (10x)m CRITICAL If the buffer contains dNTPs already,do not add additional nucleotides to the mixture
.Common oligonucleotide primers (see REAGENT SETUP for primer design
details);for example:Q T :5¢-ccagtgagcagagtgacgaggactcgagctcaagcttttttttttttttt tt-3¢
.Q O :5¢-ccagtgagcagagtgacg-3¢.Q I :5¢-gaggactcgagctcaagc-3¢
.Gene-specific oligonucleotide primers (user-specific,see REAGENT SETUP for primer design details)EQUIPMENT
.Water baths or heating blocks preset to 371,421,501,651,701and 801C .QIAquick DNA clean-up spin columns (Qiagen)or equivalent .Programmable thermal cycler REAGENT SETUP
Primer design for 5¢RACE Q T is a multipurpose primer.It contains binding sites for two,mostly non-overlapping smaller primers (Q O (Qouter)and Q I (Qinner))and an oligo dT sequence capable of annealing to the appended poly(A)tail,terminated by a non-A nucleotide to force the primer to set at t
he junction of the appended poly(A)tail and the bona fide cDNA sequence.The oligo-dT needs to be at least 17nucleotides in length to anneal at 481C.The Q O and Q I primers should be designed to work well when paired with gene-specific primers (GSPs)of standard length and GC content (18–21nucleotides,60%GC).
PROCEDURE
Reverse transcription to generate cDNA templates
1|In a sterile microcentrifuge tube,mix the following transcription components on ice:
2|In a separate sterile microcentrifuge tube,mix 0.5m l of GSP-RT primer (100ng/m l)and 1m g of poly(A)+RNA (or 5m g of total RNA)with distilled H 2O to a total volume of 12m l.Incubate at 801C for 3minutes,cool rapidly on ice,and briefly spin (centrifuge at maximum speed in a benchtop centrifuge for 5seconds at room temperature (B 251C).
m CRITICAL STEP This step disrupts the RNA secondary structure,giving the primer opportunity to anneal to its binding site in the RNA.
3|Add the RNA–primer mix to the reverse transcription components.Following this,add 1m l (200U)of
SuperScript II reverse transcriptase.Mix gently,and incubate for 1hour at 421C,and then 10minutes at 501C.
m CRITICAL STEP The reverse transcriptase is most stable at 421C,but retains activity for a short while at 501C.Raising the temperature to 501C for the final part of the reaction will result in more complete 5¢extension of the cDNA products;stalling at a GC-rich sequence can be significantly destabilized by this increase of the reaction temperature by 81C.If desired,control reactions replacing the reverse transcriptase or the GSP-RT primer with equivalent volumes of distilled water can be set up as well,for comparison purposes.
4|Inactivate the reverse transcriptase by incubating at 701C for 15minutes.Briefly spin as described in Step 2.5|Destroy the RNA template by adding 0.75m l (1.5U)of RNAse H to tube and incubating at 371C for 20minutes.
6|Dilute the reaction mixture to 400m l with Tris–EDTA buffer (10mM Tris-HCl,pH 7.5/1mM EDTA)and store at 41C.This is the 5¢end non-tailed cDNA pool.
m CRITICAL STEP Do not store at -201C,as this can cause breakage of the DNA.’PAUSE POINT The reaction mixture is stable indefinitely at 41C (refs.31,32).?TROUBLESHOOTING
p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s
/m o c .e r u t a n .w w w //:p t t h Component
Amount Final concentration Reverse transcription buffer,5x
4m l 1x
dNTPs (containing all four dNTPs,each at 10mM)1m l 1.43mM DTT,0.1M
2m l
2.88mM
a Final
concentration refers to concentration following addition of components from Steps 2and 3.
2558|VOL.1NO.6|2006|NATURE PROTOCOLS
PROTOCOL
Appending a poly(A)tail to first-strand cDNA products
7|Remove excess primer from the 5¢end non-tailed cDNA pool (obtained in Step 6above)using QIAquick spin filters or an equivalent product,according to the manufacturer’s instructions.The final volume recovered should not exceed 15m l.Adjust volume to 15m l using H 2O.
8|Add 4m l 5x tailing buffer,1.2m l CoCl 2,4m l dATP and 10U Tdt.If desired,a control reaction replacing the Tdt with an equivalent volume of distilled water can be set up as well,for comparison purposes.
9|Incubate for 5minutes at 371C and then for 5minutes at 651C.Note that the optimal temperature for the enzyme is 371C,and it is inactivated at 651C.
10|Dilute to 500m l with Tris–EDTA buffer and store at 41C.This is the 5¢end tailed cDNA pool.’PAUSE POINT The reaction mixture is stable indefinitely at 41C.?TROUBLESHOOTING
First round of amplification
11|In a sterile 0.2ml microcentrifuge tube,mix the following reagents on ice:
12|Add a 1m l aliquot of the 5¢end tailed cDNA pool (obtained in Step 10above)and 25pmols each of primers GSP1,Q O
(Fig.2b )and Q T .A control amplification replacing the template with an equivalent volume of distilled water should also be set up to ensure that the reagents are not contaminated by products that have been generated previously.
13|Mix and heat the suspension in a DNA thermal cycler for 5minutes at 981C to denature the first strand products and to activate the polymerase.Cool to 481C for 2minutes.Extend the cDNAs at 721C for 40minutes.14|Carry out 30cycles of amplification using a step program as follows:
’PAUSE POINT The reaction mixture is stable indefinitely at room temperature.
Second round of amplification
15|Dilute a portion of the amplification products from the first round 1:20in Tris–EDTA buffer.
m CRITICAL STEP A second round of amplification is required because the use of only one GSP (in combination with a universal primer that binds to all of the cDNA templates present in the starting mixture)results in a significant yield of non-specifically amplified products.The second round,which e
mploys a second GSP (again in combination with a universal primer)eliminates most to all of the non-specific products.
16|In a sterile 0.2ml microfuge tube,mix the following reagents on ice:
17|Add a 1m l aliquot of the diluted first round amplification products (obtained in Step 15above)and 25pmols each of primers GSP2and Q I (Fig.2b ).A control amplification replacing the template with an equivalent volume of distilled water should also be set up to ensure that the reagents are not contaminated by products that have been generated previously.18|Mix and heat the suspension in a DNA thermal cycler for 5minutes at 981C to denature the first strand products and activate the polymerase.
p u o r G g n i h s i l b u P e r u t a N 6002©n a t u r e p r o t o c o l s
/m o c .e r u t a n .w w w //:p t t h Component
Amount Final concentration Hercules Hot-Start polymerase buffer (10x)5m l 1x
dNTP solution (10mM)
1.0m l 200m M Hercules Hot-Start polymerase
2.5U 0.05U H 20
to 50m l
–
Cycle number Denaturation Annealing Polymerization/extension 1-2910s at 941C 10s at 52–681C 3min at 721C
30
10s at 941C
10s at 52–681C
15min at 721C,then cool to room temperature
Component
Amount
Final concentration Hercules Hot-Start polymerase buffer (10x)5m l 1x
dNTP solution (10mM)
1.0m l 200m M Hercules Hot-Start polymerase
2.5U 0.05U H 20
to 50m l
–
NATURE PROTOCOLS |VOL.1NO.6|2006|2559
PROTOCOL
豫公网安备41160202000603
站长QQ:729038198
关于我们
投诉建议