Báo cáo khoa học: Semi-nested PCR analysis of unknown tags on serial analysis of gene expression potx

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Semi-nested PCR analysis of unknown tags on serialanalysis of gene expressionWang-Jie Xu1, Qiao-Li Li1, Chen-Jiang Yao1, Zhao-Xia Wang1, Yang-Xing Zhao1andZhong-Dong Qiao1,21 College of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China2 Shanghai Institute of Medical Genetics, Shanghai Jiao Tong University, Shanghai, ChinaThe serial analysis of gene expression (SAGE) tech-nique allows the construction of a comprehensiveexpression profile, in which each mRNA is defined bya specific 14-mer [1–4]. By analyzing a short sequencetag for a transcript, SAGE significantly decreases theoverall scale of sequencing analysis and makes it possi-ble to analyze nearly all of the expressed transcriptsfrom the genome, a capability matched by no othercurrently available method [5]. Application of theSAGE technique has provided valuable information invarious biological systems [6,7]. Recently, millions ofshort cDNA sequences called SAGE tags have beencollected from human tissues through the SAGEmethod [8,9]. It has been frequently observed that alarge number of SAGE tags do not match the existingexpressed sequences upon analysis of the SAGE dataKeywordsmodified lock-docking oligo(dT); mRNA;RACE; serial analysis of gene expression(SAGE); two-step analysis of unknownSAGE tags (TSAT-PCR)CorrespondenceZ. Qiao, Shanghai Institute of MedicalGenetics, Shanghai Jiao Tong University,Shanghai, ChinaFax: +86 21 54747330Tel: +86 21 34204925E-mail: zdqiao@sjtu.edu.cn(Received 3 August 2008, revised 3September 2008, accepted 5 September2008)doi:10.1111/j.1742-4658.2008.06671.xSerial analysis of gene expression (SAGE) is a powerful technique forstudying gene expression at the genome level. However, short SAGE tagslimit the further study of related data. In this study, in order to identify agene, we developed a semi-nested PCR-based method called the two-stepanalysis of unknown SAGE tags (TSAT-PCR) to generate longer 3¢-endcDNA fragments from unknown SAGE tags. In the procedure, a modifiedlock-docking oligo(dT) with two degenerate nucleotide positions at the3¢-end was used as a reverse primer to synthesize cDNAs. Afterwards,the full-length cDNAs were amplified by PCR based on 5¢-RACE and3¢-RACE. The amplified cDNAs were then used for the subsequent two-step PCR of the TSAT-PCR process. The first-step PCR was carried out atan appropriately low annealing temperature; a SAGE tag-specific primerwas used as the sense primer, and an 18 bp sequence (universal primer I)located at the 5¢-reverse primer end was used as the antisense primer. After15–20 PCR cycles, the 3¢-end cDNA fragments containing the tag could beenriched, and the PCR products could be used as templates for the second-step PCR to obtain the specific products. The second-step PCR was per-formed with a SAGE tag-specific primer and a 22-bp sequence (universalprimer II) upstream of universal primer I at the 5¢-reverse primer with ahigh annealing temperature. With our innovative TSAT-PCR method, wecould easily obtain specific PCR products covering SAGE from those tran-scripts, especially low-abundance transcripts. It can be used as a method toidentify genes expressed in different cell types.AbbreviationsGLGI, generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification; PLF, primary library forwardprimer; PLR, primary library reverse primer; RAST-PCR, rapid reverse transcription–PCR analysis of unknown serial analysis of geneexpression tags; rSAGE, reverse serial analysis of gene expression; SAGE, serial analysis of gene expression; TSAT-PCR, two-step analysisof unknown SAGE tags; UP-I, universal primer I; UP-II, universal primer II.5422 FEBS Journal 275 (2008) 5422–5428 ª 2008 The Authors Journal compilation ª 2008 FEBS[10,11]. It is possible, then, that the unmatched SAGEtags originating from potentially novel transcripts ornovel genes are unidentified in the human genome.We have constructed a SAGE library on humanspermatozoa in which we obtained more than 2500unique tags. Of these, 54 were considered to be high-frequency tags, and no homology could be found inthe GenBank database [12]. Therefore, those tagsmight represent unidentified genes. However, there wasa major problem when the SAGE tag sequence wasapplied to the process of gene identification. Owing tothe short length of SAGE tag sequences, it became dif-ficult to produce the 3¢-longer cDNA fragments andeven whole cDNA sequences by PCR, which affectedfurther studies on SAGE data. Moreover, the shorttag has hindered the application of SAGE to the vastmajority of eukaryotes, including expressed sequencetags and genome sequences without sufficient genomicresources [13].In order to solve this problem, we have developeda technique called the two-step analysis of unknownSAGE tags (TSAT-PCR) to generate the 3¢-longercDNA ends. The three key points of our method areas follows: first, it uses a modified lock-dockingoligo(dT) primer, with two degenerate nucleotidepositions at the 3¢-end, as a reverse primer to syn-thesize the first-strand cDNA; second, the primarycDNAs were enriched by PCR, and then served astemplates for the subsequent TSAT-PCR experiment;and third, the semi-nested PCR principle was usedas a reference in designing the two-step PCR methodin order to obtain the 3¢-end cDNA tag-specificfragments. Currently, we have successfully used thisprocedure to test and analyze 11 of the 54unmatched SAGE tags.Results and DiscussionEnrichment of cDNA templateOwing to RACE technology, we could now amplifyfull-length cDNAs to generate enough templates forthe subsequent PCR, especially a few low-abundancecDNAs (Fig. 1A). In this study, the amplification ofcDNAs was carried out as follows: first, owing totwo degenerate nucleotide positions at the 3¢-end ofthe modified oligo(dT) primer in the RT-PCR pro-cess, these nucleotides position the primer at the startof the poly(A)+tail, thereby eliminating the 3¢-heter-ogeneity inherent in conventional oligo(dT) priming[14]. As the PrimeScript Reverse Transcriptase exhib-ited terminal transferase activity upon reaching theend of an RNA template, it added three to five resi-dues (predominantly dC) to the 3¢-end of the first-strand cDNA. The 5¢-cap oligonucleotide contained aterminal stretch of G residues that annealed to thedC-rich cDNA tail and served as an extended tem-plate for reverse transcription. In the subsequentPCR process, the reverse transcription product abovewas used as template. Primary library forward primer(PLF) and primary library reverse primer (PLR)paired with the 5¢-end and 3¢-end of all cDNAs,respectively, and after 25 cycles, the entire cDNAswere largely amplified for the next experiment.Figure 2 shows the amplified cDNAs. As can beseen, the length of the smear is distributed from aboutmRNAmRNANBAAAAAAA-3′NBAAAAAAA-3′NBAAAAAAAModified oligo (dT)NVTTTTTTTNBAAAAAAANVTTTTTTTNBAAAAAAANVTTTTTTTNBAAAAAAANVTTTTTTTNBAAAAAAANVTTTTTTT16161616161616165′5′5′-cap oligoNVTTTTTTTNVTTTTTTTNBAAAAAAANVTTTTTTTGGGCCCGGGCCCGGGCCCGGGCCCGGGCCCAnneal first strandPrimer to mRNAcDNA firststrand synthesisModified oligo (dT)Tag-specific primerUP-IThe 2nd PCRThe 1st PCRUP-IIUP-IIPLFPLRGGATCCGGATCCGGATCCcDNAs synthesiscDNA libraryABFig. 1. Detailed mechanism of the amplification of the whole cDNAs and the TSAT-PCR technique. (A) In this process, double-strandedcDNAs synthesized by modified lock-docking oligo(dT) and 5¢-cap oligonucleotides were used for PCR. During the PCR process, PLF and PLRwere used as sense primer and antisense primer, respectively, to amplify the cDNAs. (B) The procedure involved two PCR reactions. The firstPCR reaction was performed with a tag-specific primer containing a SAGE tag sequence and an 18 bp primer (UP-I) located at the 5¢-reverseprimer end. The first PCR product was then used as the template for the second PCR reaction. The tag-specific primer and a 22-bp primer(UP-II) located near UP-I located at the 5¢-reverse primer were used as the sense primer and the antisense primer, respectively.W J. Xu et al. New method of 3¢-end amplification from SAGE tagsFEBS Journal 275 (2008) 5422–5428 ª 2008 The Authors Journal compilation ª 2008 FEBS 5423100 bp to over 2 kb, and is mostly focused on the 0.3–1 kb range. The results demonstrate that high-abun-dance genes are not very variable in terms of length,as they mostly concentrate on a narrow span (0.3–1 kb). Aside from the range, we can see that there area few low-abundance genes that are either very long(50 kb) or short (50 bp). It seems that the smear of thegenes did not become obvious because of their lowabundance or short extension time in the PCR, orboth.TSAT-PCR general strategyThe amplified cDNAs served as primary templates forTSAT-PCR, as illustrated in Fig. 1B. The antisenseprimers [PLR, universal primer I (UP-I) and universalprimer II (UP-II)] were all designed from the sequenceof the modified oligo(dT) primer. The three primersshared some overlap with each other and their lengthwas different considering the consistency of theirequivalent sense primers (Fig. 3). Both UP-I andUP-II were used as nested primers in the TSAT-PCRreactions. The TSAT-PCR technique was developedfrom the principle of nested PCR, and the procedureincluded a two step-PCR reaction. For 15–20 cycles ofthe first PCR, an appropriately low annealing tempera-ture (about 55 °C) was used, a SAGE tag-specificprimer and UP-I. As a result, the 3¢-end cDNA frag-ments containing the tag could be enriched while somenonspecific products were also generated simulta-neously, and then the PCR products could be usedas templates for the second-step PCR to obtain thespecific products. The second-step PCR was performedwith a SAGE tag-specific primer and a nested primer(UP-II) at a high annealing temperature (‡ 60 °C).Afterwards, the specific products corresponding to tagscould be amplified.Amplification of longer sequences fromSAGE tagsTo test the TSAT-PCR procedure, we chose five tagscorresponding to known genes, as well as 11 different-abundance tags corresponding to unknown genes, allidentified in SAGE analysis of human spermatozoa(Table 1). Among the 16 tags, tag 4, A and E wereused as representatives of low-frequency genes in orderto help us determine whether or not the processworked on low-frequency tags. Upon application ofthe TSAT-PCR method, we obtained the PCR prod-ucts (Fig. 4) of all tags tested using the standard PCRcondition (first PCR, 94 °C for 30 s, 55 °C for 30 sand 72 °C for 30 s for 15 cycles; second PCR, 94 °Cfor 30 s, 60 °C for 30 s and 72 °C for 30 s for 25cycles). The PCR products were electrophoresedthrough a 2.0% agarose gel, and cloned into a plasmidvector for sequencing analysis. As compared with theothers, tag 1, 2, 3, 4 and A displayed very weak PCRbands in the agarose gel, especially the two low-frequency tags (Fig. 4). Aside from this, there werealso two clear bands in the PCR product of no. 10.We further optimized the PCR annealing tempera-ture, as well as the cycle number, for each of theweak-band tags. Moreover, these bands were obviouslyclearer than the pervious ones (data not shown). Wethen verified whether or not each PCR product indeedrepresented a sequence downstream of the most 3¢NlaIII site in the full-length cDNA by analyzing theM123530 bp1584 bp947 bp564 bpFig. 2. PCR amplification of the full-length cDNAs. The cDNAswere amplified with PLF and PLR. M: kDNA ⁄ HindIII + EcoRI mar-ker. Lane 1: amplified full-length cDNAs. Lane 2: glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was used as control.Fig. 3. The sequences and relationships of the primers [modified oligo(dT), PLR, UP-I and UP-II] discussed in this article.New method of 3¢-end amplification from SAGE tags W J. Xu et al.5424 FEBS Journal 275 (2008) 5422–5428 ª 2008 The Authors Journal compilation ª 2008 FEBSsequences of the products. If the tag sequence waspresented at the predicted location, no NlaIII sitewould be present in the sequence of the obtained PCRproduct, whereas the PCR product would include theoligo(dT16) sequence. All PCR products were clonedand sequenced successfully (Table S1). Through analy-sis of the sequencing result, we identified 16 of 17 PCRproducts (Figs 4 and 5) that met the standard men-tioned above. This indicates that the 16 PCR productsrepresented a sequence downstream of the most 3¢NlaIII restriction site. In contrast, the remaining PCRproduct was a large size band of the no. 10 product, inwhich sequences of UP-II and oligo(dT16) were notfound, although the tag-specific primer was foundonly in its sequence. This meant that the PCR productwas amplified by PCR using only a single primer (thetag-specific primer no. 10). Sequencing could onlydetermine the single primer-prone product. Thesequencing results (Table 1) were analyzed using theblast program of the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST/). Among the five fragmentscontaining known tags (Table 1), four sequences corre-sponding to the tags A, B, C and E were matched tothe 3¢-cDNA of genes predicted by Zhao based on thespermatozoa SAGE tags [12], whereas no. D was notmatched to the gene (Hs. 436980). The reason for thiswas further investigated, and it was found that theno. D tag could not represent the gene (Hs. 436980),because seven NlaIII (CATG) sites were foundbetween the site of the no. tag D tag and a poly(dA)among the cDNA of the gene (Hs. 436980). The blastresults of another 11 sequences in the GenBankTable 1. Overview of all tags analyzed with the TSAT-PCR technique. The sequences from nos. 1 and 7 matched a single sequence. No. 11matched multiclusters. The rest of the sequences did not match any clusters.Tag Tag sequence UniGene ID AbundancePCR productsize (bp)Presenceof NlaIII sitePresenceof oligo(dT) Blast resultsA ACTTACCTGC Hs. 431668 6 89 No Yes ConsistencyB GCGTGCCTGC Hs. 372658 302 211 No Yes ConsistencyC GCCCCTGCGC Hs. 435464 214 217 No Yes ConsistencyD GTGACCACGG Hs. 436980 126 189 No Yes InconsistencyE GTGGCACACG Hs. 34114 5 192 No Yes Consistency1 AACGAGGAAT – 84 254 No Yes AK0273222 GTAAGTGTAC – 44 97 No Yes Unmatched3 AGAGGTGTAG – 30 232 No Yes Unmatched4 TTGCCAACAC – 4 94 No Yes Unmatched5 GAAGTCGGAA – 58 101 No Yes Unmatched6 GCCGTTCTTA – 21 198 No Yes Unmatched7 ATTAAGAGGG – 16 165 No Yes NR_0032868 ATGCCTGTAG – 16 182 No Yes Mismatch9 GCCTTGTTCA – 13 184 No Yes Unmatched10 TTCTCAATGA – 10 274 No Yes Unmatched317 No No –a11 CCCATCGTCC – 9 123 No Yes BC010864BC021246BC013387AY211920BC092442aSingle-prime PCR product.500 bpM 1 2 3 4 5 6 7 8 9 10 11 a b c d e 400 bp300 bp200 bp100 bpFig. 4. TSAT-PCR analysis of 16 tags. Lanes 1–11 were unknown SAGE tags corresponding to tags 1–11 in Table 1. Lanes a, b, c, dand e were known SAGE tags corresponding to tags A, B, C, D and E in Table 1. TSAT-PCR was performed as described in Results andDiscussion.W J. Xu et al. New method of 3¢-end amplification from SAGE tagsFEBS Journal 275 (2008) 5422–5428 ª 2008 The Authors Journal compilation ª 2008 FEBS 5425database (refseq_rna: reference mRNA sequence andexpressed sequence tags) revealed several cases(Table 1): match, multimatch, unmatch and mismatch.The corresponding accession numbers of matched andmultimatched sequences are given in Table 1. No. 8was defined as a mismatch, because the blast resultshowed that the site of the tag did not exactly matchsequences in the GenBank database, due to nonspecificamplification. The genes corresponding to the matchedsequences (corresponding to tags A and E) areHs. 431668 (COX6B1, cytochrome c oxidase subunitVib polypeptide 1) and Hs. 34114 [ATP1A2, ATPase,Na+⁄ K+-transporting, a2(+) polypeptide], which arerelated to energy production for motility of the humanspermatozoa. Hs. 372658, corresponding to no. B, is agene coding for spermatogenesis-related protein 7,which could take part in spermatogenesis. The rest ofthe genes corresponding to tags C, 1 and 7 areHs. 435464 (Homo sapiens neuritin 1-like), AK027322(highly similar to signal recognition particle 68 kDaprotein), and NR_003286 (Homo sapiens 18S ribo-somal RNA). Currently, as little is known of the func-tion of mRNAs in human spermatozoa, it was difficultto estimate whether the rest of the genes were relatedto the function of human spermatozoa, or just retainedduring spermatogenesis. For the unmatched sequencesand multimatched sequences, the 5¢-RACE experimentshould be carried out to obtain its full-length cDNAsequences and to determine whether the sequencesrepresent new genes.During the course of our research on the SAGEdata of the human spermatozoa, we became aware thatother methods [rapid reverse transcription–PCR analy-sis of unknown SAGE tags (RAST-PCR) [15], genera-tion of longer cDNA fragments from SAGE tags forgene identification (GLGI) [16] and reverse SAGE(rSAGE) [17]] hardly generate the 3¢-fragmentsequences of these unmatched tags. Although GLGI ismore effective than RAST-PCR [17], the antisense pri-mer in GLGI is only composed of oligo(dT), so therigorous PCR conditions, the Mg2+concentration, thenumber of PCR cycles and the annealing temperaturewould be optimized for each SAGE tag. In experi-ments, we often encountered nonspecific amplificationor multiple fragments, and met difficulties in amplify-ing the product of low-frequency tags, due to the shortantisense primer. The rSAGE method was derivedfrom SAGE, and many steps and reagents are sharedby these two protocols. However, step 4 (linker liga-tion) in the rSAGE protocol does not avoid self-ligation of the cDNA, and the self-ligation would leadto smearing in the following PCR amplification. Inaddition, the method requires more initial total RNAand poly(A)+than SAGE, because of the loss ofRNA in each step. Thus, the demand for RNArestricts the application of this method during the lowtotal RNA experiment, as each human spermatozoonis estimated to contain just 0.015 pg of total RNA[18], only 1 ⁄ 600 of the amount of somatic total RNA.To avoid this problem, we have used semi-nestedPCR to improve the specific amplification, and devel-oped the method called TSAT-PCR. Using the condi-tions described in that article [17], we compared the twomethods with six tags and obtained the results that weexpected (Fig. 5). The bands obtained with TSAT-PCRare obviously clearer than those obtained within GLGI;moreover, the tags (4, A and E) with low abundance(< 6) were all obtained with TSAT-PCR.In comparison with other methods, ours is able toamplify our target PCR products from low-abundancetranscripts. Also, the method needs a lower initialamount of mRNA than the with others. Furthermore,our method possesses the advantages of being simple,rapid, low in cost, and highly efficient. We have dem-onstrated that we could obtain a clear band of PCRproducts for each case, as well as enough full-lengthcDNAs as PCR templates for subsequent experimentsthrough the novel PCR amplification method describedabove.Although the improved version of SAGE can gener-ate tags with lengths of 21 bases [19] and 26 bases [13],which theoretically can be uniquely assigned to a single500 bp400 bp300 bp200 bp100 bpM E C B A 10 4 E C B A 10 4 TSAT-PCR GLGIFig. 5. Comparison between GLGI and TSAT-PCR. A set of six SAGE tags was chosen for the analysis. Among the six tags, three tags(4, A and E) with low abundance (< 6) were examined. The same RNA from human spermatozoa and sense primers was used for bothmethods. The conditions used for GLGI followed the procedures described in [16].New method of 3¢-end amplification from SAGE tags W J. Xu et al.5426 FEBS Journal 275 (2008) 5422–5428 ª 2008 The Authors Journal compilation ª 2008 FEBSgenomic position [20], there still exists a much earlierSAGE database constructed with the use of theconventional SAGE technique, which consists ofshorter tags (14 bp). Converting short tags to 3¢-longercDNA is a key step and a breakthrough for furtherstudies on SAGE data. Our method would help SAGEto become a high-throughput technique that could bewidely applied to gene expression.In summary, the study could be applied to furtheranalyses of SAGE data gathered from humans andsome eukaryotic species. Our approach has severalimportant advantages, such he following: (a) it canobtain enough full-length cDNA templates for sub-sequent experiments, such as 5¢-RACE, 3¢-RACE andnorthern blotting, among others; (b) it can convertshort SAGE tag sequences into 3¢-complementaryDNAs; (c) it can obtain full-length DNA sequencescontaining specific tags from mRNA transcripts, espe-cially low-abundance mRNA transcripts, through thecombined application of TSAT-PCR and 5¢-RACE;and (d) it can identify novel genes from SAGE dataand confirm the existence of exons predicted by bio-informatic tools in genomic sequences.Experimental proceduresTag sequencesIn our SAGE library generated from human spermatozoa,each tag was homologously screened in the Unigene data-base (http://www.ncbi.nlm.nih.gov/SAGE/SAGEtag.cgi?tag)to identify its respective match. We chose 16 SAGE tags,including four tags corresponding to known genes, whichserved as a positive control for this experiment, and 11 dif-ferent-abundance tags from the 54 unmatched tags corre-sponding to unknown genes.RNA samples and cDNA synthesisTotal RNA of purified spermatozoa was extracted usingTrizol RNA isolation reagent (Invitrogen, Carlsbad, CA,USA), according to the manufacturer’s protocol (http://www.invitrogen.com/content/sfs/manuals/10296010.pdf). Thequantity of extracted RNA was determined by UV absorp-tion. Meanwhile, cDNAs were generated with a modifiedRACE method through the PrimeScript Reverse Transcrip-tase (TaKaRa, Dalian, China), following the manufacturer’sinstructions. Briefly, two kinds of primers were added in theRT-PCR reaction: one was the modified oligo(dT) primer(5¢-CCAGA CACTATGCTCATACGACGCAG-T16-VN-3¢;N =A,C,G,orT;V = A, G, or C), which was used as areverse transcription primer to generate the first-strandcDNA; and the other was the 5¢-cap oligonucleotide primer(5¢-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3¢),which annealed to the dC-rich cDNA tail and served as anextended template for reverse transcription. Thus, a set offull-length cDNAs can now serve as a primary library ofspermatozoa cDNAs to be used for further studies.Amplification of primary libraryThe full-length cDNAs in spermatozoa were amplified byPCR with the use of Takara Ex Taq Hot Start Version(TaKaRa), with the primary library sequences serving as thetemplate. Briefly, PLF (5¢-AAGCAGTGGTATCAACGCAGAGT-3¢) was used as the sense primer, and was located atthe 5¢-end of all cDNAs generated from the 5¢-cap oligonu-cleotide primer. Meanwhile, PLR, which used the sequence(5¢-CCAGACACTATGCTCATACGACG-3¢) in the 3¢-endsof all cDNAs incorporated from the reverse transcriptionprimer, was used as the antisense primer in the PCR. ThePCR program consisted of 25 cycles of 94 °C for 30 s, 66 °Cfor 30 s and 72 °C for 3 min. The final extension step con-sisted of 72 °C for 5 min. Ten microliters of the PCR productwas checked by 1.2% agarose gel electrophoresis.TSAT-PCRThe amplified primary library was diluted 103-fold withsterile H2O for TSAT-PCR analyses. A 1-lL aliquot wasdirectly used as a template for the first PCR amplificationwith the tag-specific primer (5¢-GGATCCXXXXXXXXXX,X represents each tag) and UP-I (5¢-CCAGACACTATGCTCATA-3¢). The reaction was then carried out for 15cycles with the following conditions: 94 °C for 30 s, 53–55 °C for 30 s and 72 °C for 30 s extension with TaKaRaEx Taq (TaKaRa), using a Bio-Rad Cycler (Bio-Rad, Her-cules, CA, USA). The resulting PCR product was diluted103-fold with sterile H2O, and a 1 lL aliquot was used as atemplate for the second nested PCR amplification with thetag-specific primer and UP-II (5¢-CACTATGCTCATACGACGCAGT-3¢) with the following conditions: 25–30cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s,using TaKaRa Ex Taq (TaKaRa).DNA cloning and sequencingThe PCR products were cloned into pT19G-T vector (Gen-eray Biotech, Shanghai, China). Positive clones werescreened by PCR with M13 reverse and M13 forward(220 bp) primers while located in the vector; sequencingreactions were performed by Sanny Bio-Tech (Shanghai,China).AcknowledgementsThis work was supported by Shanghai LeadingAcademic Discipline Project (B205).W J. Xu et al. New method of 3¢-end amplification from SAGE tagsFEBS Journal 275 (2008) 5422–5428 ª 2008 The Authors Journal compilation ª 2008 FEBS 5427References1 Velculescu VE, Zhang L, Vogelstein B & Kinzler KW(1995) Serial analysis of gene expression. 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The amplified longer cDNA sequences.This supplementary material can be found in theonline version of this article.Please note: Wiley-Blackwell is not responsible forthe content or functionality of any supplementarymaterials supplied by the authors. Any queries (otherthan missing material) should be directed to thecorresponding author for the article.New method of 3¢-end amplification from SAGE tags W J. Xu et al.5428 FEBS Journal 275 (2008) 5422–5428 ª 2008 The Authors Journal compilation ª 2008 FEBS . analysis of gene expression tags; rSAGE, reverse serial analysis of gene expression; SAGE, serial analysis of gene expression; TSAT -PCR, two-step analysis of unknown. toidentify genes expressed in different cell types.AbbreviationsGLGI, generation of longer cDNA fragments from serial analysis of gene expression tags for gene
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