A strategy of genome library from single cells. 

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... should be applied in single-cell studies. At present, there are two strategies to employ genome information amplification: mRNA amplification (aRNA) and PCR-based cDNA amplification. The aRNA procedure begins with total RNA or poly(A) RNA that is reverse transcribed using an oligo (dT) primer containing a T7 RNA polymerase promoter sequence. After first-strand synthesis, the reaction is treated with RNase H to fragment the mRNA. These fragments serve as primers during a second-strand synthesis reaction that produces a double-stranded DNA template for transcription. rRNA, mRNA fragments and primers are removed before using the cDNA template to produce linearly amplified aRNA. The amplification yields can reach 1000-5000 fold following two rounds of in vitro transcription (Figure 2-I). RNA amplification and is commercially available and has been increasingly reported in gene expression studies (Eberwine, 1996). PCR-based amplification has two protocols: specific profile and global profile applications. Specific profile methods such as rtPCR or multiplex rtPCR reactions are sensitive at the single-cell level, especially in nested PCR. Because the genes studied this way are pre-selected, it can only be applied to known genes. In order to overcome the problem, global PCR-based approaches have been developed in genomic analysis. One approach is homomeric tailings, and another is 3 ́-(3-primer-end) amplification (TPEA). The former (Figure 2-II) (Toellner et al. 1996) uses terminal deoxynucleotide transferase-generated homomeric 3’ tails to the first-strand cDNA. After rtPCR and 3’ tailing addition and PCR amplification, it has been applied to the analysis of single-cell global gene expression. Even though homomeric tailings can be used effectively in global profile analysis, many of the cDNA copies are not full length and shorter cDNAs are preferentially amplified. 3-primer-end- amplification (TPEA, Figure 2-III) (Dixon et al. 1998) is a partially randomized amplification of mRNA using an oligo-dT primer together with a 5’ primer containing a random pentamer. It can enable the detection of both high- and low-abundance mRNA transcripts from single cells. Because TPEA also has a 3’ bias, full-length cloning is more difficult. Moreover, only 40 to 50 genes can be analyzed in samples derived from single cells. We have also developed a more facile strategy to screen the genome at the single cell level. To illustrate, three techniques (RNA directly from cell lysis, randomized primer design as differential display and single cell genome cloned into plasmids) are simultaneously combined (Li et al. 2000). Following cell lysis and reverse transcription PCR, a 3’ end oligo (dT) n primer and a set of 5’ end arbitrary primers (both containing restriction enzyme terminals) are used in an amplification by PCR. After double digestion, the genome from a small number of cells is introduced into plasmids and transformed into cells. As Figure 2 and Figure 3 indicate, subtractive hybridization from a reference cell genome is employed in the modified method so that artefacts of cDNA amplification from test cells are minimized (Li et al. 2002). The technique has been used in genome expression analysis from 10 to 100 cells. The advantage is that the expression results are very sensitive and accurate because they exclude problems of artefacts. The disadvantage is similar to TPEA, that is, expression has a 3’ bias (Figure 2-IV). Over the past two years, as aRNA techniques have been developed, cDNA detection sensitivity has significantly increased so that we can use either cells from microdissection or single cells obtained from culture to analyze their genome expression (Figure 3) (Zhang et al. 2004). Protein signal amplification from a single-cell . Traditional methodologies for protein detection and quantification include two-dimensional gel electrophoresis, mass spectrometry, and antibody (Ab) binding. As we discussed previously, the application of these traditional proteomics-oriented technologies at the single-cell level has been limited because each methodology needs relatively large amounts of tissue. The development of protein arrays using antibody-binding technology has presented a new opportunity to study protein expression at the single cell level. Recently, two protein array techniques have rapidly been extended. The first is the use of intact antibodies, antibody fragments (single-chain fragment variable (ScFv) fragments) or exocyclic peptide-based complementarity determining region (CDR) subunits as antigen detectors. The second is the Ab used as amplification signals. In order to detect protein and peptide molecules, several Ab signal amplifications have been successfully employed in order to improve sensitivity. For example, immuno-PCR and T7 RNA amplification have been reported in several journals. In the former, the PCR technology is combined ...

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... should be applied in single-cell studies. At present, there are two strategies to employ genome information amplification: mRNA amplification (aRNA) and PCR-based cDNA amplification. The aRNA procedure begins with total RNA or poly(A) RNA that is reverse transcribed using an oligo (dT) primer containing a T7 RNA polymerase promoter sequence. After first-strand synthesis, the reaction is treated with RNase H to fragment the mRNA. These fragments serve as primers during a second-strand synthesis reaction that produces a double-stranded DNA template for transcription. rRNA, mRNA fragments and primers are removed before using the cDNA template to produce linearly amplified aRNA. The amplification yields can reach 1000-5000 fold following two rounds of in vitro transcription (Figure 2-I). RNA amplification and is commercially available and has been increasingly reported in gene expression studies (Eberwine, 1996). PCR-based amplification has two protocols: specific profile and global profile applications. Specific profile methods such as rtPCR or multiplex rtPCR reactions are sensitive at the single-cell level, especially in nested PCR. Because the genes studied this way are pre-selected, it can only be applied to known genes. In order to overcome the problem, global PCR-based approaches have been developed in genomic analysis. One approach is homomeric tailings, and another is 3 ́-(3-primer-end) amplification (TPEA). The former (Figure 2-II) (Toellner et al. 1996) uses terminal deoxynucleotide transferase-generated homomeric 3’ tails to the first-strand cDNA. After rtPCR and 3’ tailing addition and PCR amplification, it has been applied to the analysis of single-cell global gene expression. Even though homomeric tailings can be used effectively in global profile analysis, many of the cDNA copies are not full length and shorter cDNAs are preferentially amplified. 3-primer-end- amplification (TPEA, Figure 2-III) (Dixon et al. 1998) is a partially randomized amplification of mRNA using an oligo-dT primer together with a 5’ primer containing a random pentamer. It can enable the detection of both high- and low-abundance mRNA transcripts from single cells. Because TPEA also has a 3’ bias, full-length cloning is more difficult. Moreover, only 40 to 50 genes can be analyzed in samples derived from single cells. We have also developed a more facile strategy to screen the genome at the single cell level. To illustrate, three techniques (RNA directly from cell lysis, randomized primer design as differential display and single cell genome cloned into plasmids) are simultaneously combined (Li et al. 2000). Following cell lysis and reverse transcription PCR, a 3’ end oligo (dT) n primer and a set of 5’ end arbitrary primers (both containing restriction enzyme terminals) are used in an amplification by PCR. After double digestion, the genome from a small number of cells is introduced into plasmids and transformed into cells. As Figure 2 and Figure 3 indicate, subtractive hybridization from a reference cell genome is employed in the modified method so that artefacts of cDNA amplification from test cells are minimized (Li et al. 2002). The technique has been used in genome expression analysis from 10 to 100 cells. The advantage is that the expression results are very sensitive and accurate because they exclude problems of artefacts. The disadvantage is similar to TPEA, that is, expression has a 3’ bias (Figure 2-IV). Over the past two years, as aRNA techniques have been developed, cDNA detection sensitivity has significantly increased so that we can use either cells from microdissection or single cells obtained from culture to analyze their genome expression (Figure 3) (Zhang et al. 2004). Protein signal amplification from a single-cell . Traditional methodologies for protein detection and quantification include two-dimensional gel electrophoresis, mass spectrometry, and antibody (Ab) binding. As we discussed previously, the application of these traditional proteomics-oriented technologies at the single-cell level has been limited because each methodology needs relatively large amounts of tissue. The development of protein arrays using antibody-binding technology has presented a new opportunity to study protein expression at the single cell level. Recently, two protein array techniques have rapidly been extended. The first is the use of intact antibodies, antibody fragments (single-chain fragment variable (ScFv) fragments) or exocyclic peptide-based complementarity determining region (CDR) subunits as antigen detectors. The second is the Ab used as amplification signals. In order to detect protein and peptide molecules, several Ab signal amplifications have been successfully employed in order to improve sensitivity. For example, immuno-PCR and T7 RNA amplification have been reported in several journals. In the former, the PCR technology is combined ...

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... immuno-detection methods as shown in Figure 4(A) (McKie et al. 2002). Streptavidin is added to a biotinylated Ab-antigen complex whereupon a known biotinylated-DNA fragment is added, resulting in the formation of a specific antigen-Ab-DNA conjugate. The attached marker DNA can be amplified by PCR with appropriate primers. Some results have shown that ~10 5 fold increase in sensitivity over an alkaline phosphatase- conjugated ELISA is obtained. This approach, with slight modifications, has been used to detect a variety of antigens, including a human protooncogene protein and tumour necrosis factor α . Although the immuno-PCR technique has some advantages over traditional methods of protein detection, such as increase in sensitivity, there still exist several notable limitations to its use. One of the major limitations of immuno-PCR lies in the nonlinear amplification ability of PCR, which limits this technique as a quantitative detection method. Some of these problems have been overcome with a relatively isothermal rolling circle DNA amplification technique (RCA). As demonstrated in Figure 4(B) (Zhang et al. 2001), T7 RNA amplification resolves these problems and shows a linear relationship between protein expression and an expression indicator such as luciferase. Single-cell gene expression analysis can be carried out both at the specific profile and global genome profile. In situ hybridization and rtPCR belong to the specific profile. In situ PCR combined with immunohistochemical detection is frequently used as a measurement of single-cell gene activity (Gey et al. 1999). Multiplex rtPCR is also effective for observing gene expression at the single-cell level (Hahn et al. 2002). At present, rtPCR using real-time detection of PCR products can quantify gene expression at the single- cell level with reduced risk for artefacts resulting from contamination or illegitimate transcript amplification (Liss, 2002). However, because the primers are pre-selected, expression profiles will not contain previously unreported transcripts or novel sequences. Global genome profile expression analysis at the single-cell level holds new promise to analyze disease pathogenesis and tumorigenesis. At present, four techniques are utilized to advance the global genome profile of a single cell (in addition to the previously described specific profiles such as in situ hybridization and multiplex rtPCR). These global genome profiles include differential display, subtractive cloning, microarray, and protein array. As shown in Figure 2 and Figure 3, differential display and subtractive cloning can be employed with a small number of cells in which the resolution is from one cell to 10 4 cells (Chen and Talmage, 1999). Because both of these methods may have an artefact contamination after amplification, it may result in variable genome expression at the single-cell level. As discussed above, we introduced a strategy combining amplifying RNA, randomized primers (with restriction terminals for cloning into plasmid) and subtractive hybridization (for eliminating some artefacts), which has successfully been used in genome expression at single-cell level. Although our method still has a problem in screening the genome, that is, some 3’ bias, after sequencing the genome at 3’ terminal fragment, GenBank analysis can allow us to eventually determine full-length genome prediction (Li et al. 2000; Li et al. 2002; Zhang et al. 2004). Here, some explanation and details shown as above (such as including A: designs of random primers with restriction enzyme site, B: cDNA amplification, C: cloning into plasmid and storage of library and D: subtractive hybridization using reference cell cDNA), we illustrate the basic protocol ...