WO2002092774A2

WO2002092774A2 - Replicase cycling reaction amplification

Info

Publication number: WO2002092774A2
Application number: PCT/US2002/015426
Authority: WO
Inventors: Shi-Lung Lin; Henry H. Ji
Original assignee: Shi-Lung Lin; Ji Henry H
Priority date: 2001-05-14
Filing date: 2002-05-14
Publication date: 2002-11-21
Also published as: WO2002092774A3; AU2002314782A1

Abstract

There is disclosed a process for amplifying RNA sequences in a simple and rapid procedure. Specifically, there is disclosed a process, compounds and kits for replicase cycling reactions for amplifying RNA sequences from a wide variety of sources such as cellular RNAs or genomes

Description

REPLICASE CYCLING REACTION AMPLIFICATION Technical Field of The Invention

The present invention provides a process for amplifying RNA sequences in a simple and rapid procedure. Specifically, the present invention provides a process, compounds and kits for replicase cycling reactions for amplifying RNA sequences from a wide variety of sources such as cellular RNAs and/or genomes. Background of the Invention

The ability to amplify nucleic acid sequences from cells has permitted the molecular investigations of intracellular gene and/or genome status under certain special conditions, such as pathogenesis, mutation, treatment processing and developmental control. Traditionally, nucleic acid sequences are isolated from genomic DNAs and/or cellular RNAs (Sambrook et al., "Molecular Cloning, 2nd Edition ", pp8.11-8.35 (1989)). However, the tedious procedures of extraction, purification and cloning usually fails to maintain the completeness of all nucleic acid sequences, and results in a significant loss of rare DNA or RNA populations. Moreover, another problem has been the requirement of bulk tissue samples for nucleic acid extraction. Current genetic/genomic analysis and molecular diagnosis rely on relatively pure sample collections with high throughput and high-resolution capacity. Unfortunately, it is difficult if not impossible to collect enough pure or homogeneous samples for traditional extraction methods due to the fast degradation rate of nucleic acid sequences, especially messenger RNAs (mRNA).

On the other hand, the generation of amplified complementary DNA (cDNA) products by polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR) has become the most common way among current nucleic acid amplification methods. Prior art attempts at amplifying DNA sequences with PCR, such as United States Patent No. 4,683,202 and 4,965,188 to Mullis, and with RT-PCR, such as United States Patent No. 5,817,465 to Mallet, uses reverse transcriptase and DNA polymerases to generate cDNA products based on a thermal cycling strategy. Although these PCR-based methods successfully produce high quantity of cDNAs, the low fidelity of these products is usually a problem that results from the high mis-reading rate of most thermostable DNA polymerases. Moreover, the preferential amplification of nonspecific products occurs very often, incurring inevitable bias and difficulty in genetic/genomic analysis (Sambrook et al., supra; Lin et al., Nucleic Acid Res. 27:4585- 4589 (1999)). These disadvantages diminish the feasibility of PCR-based methods in current DNA or oligonucleotide microarray analysis.

The generation of antisense RNA (aRNA) or complementary RNA (cRNA) sequences with in vitro transcription reaction has provided for linear amplification of nucleic acid sequences from limited cells (Van Gelder et al., Proc. Natl. Acad. Sci. USA 87:1663-1667 (1990); Compton, Nature 350:91-92 (1991)). Prior art attempts at aRNA/cRNA amplification, such as United States Patent No. 5,514,545 to Eberwine, United States Patent No. 5,888,779 to Kacian and United States Patent No. 6,197,554 to Lin., uses reverse transcription to incorporate an RNA promoter into a cDNA template for further transcriptional amplification of aRNAs/cRNAs. Although these aRNA/cRNA amplification methods lead to the identification of some useful RNA markers for disease detection, the rare mRNA-representative copies are not preserved due to the low affinity of oligo (dT)-promoter primers used in these methods (O'Dell et al., BioTechniques 25:566-570 (1998)). Therefore, there is a need in the art to find better RNA amplification methods that will be able to detect rare RNA species.

In summary, it is desirable to have a fast, simple and reliable amplification process for generating amplifiable nucleic acid sequences without the drawbacks of PCR-based and cRNA-dependent methodologies. The amplified nucleic acid products may be used, for example, for screening differential gene sequences, for facilitating microarray analysis, for searching for functional domains in genes and/or genomes, for producing synthetic peptides in vitro, and even for designing diagnostic or therapeutic products. Summary of the Invention

The present invention provides a replicase cycling process for amplifying an RNA source, comprising:

(a) providing a source RNA material for amplification; and

(b) incubating the source RNA material with a solution comprising (1) a capture molecule, (2) a plurality of ribonucleotides (rNTPs), and (3) a replicase or RNA-dependent RNA polymerase enzyme activity, wherein the capture molecule has a formula I: SLC site - linker moiety - HO« I or a cap-capture molecule having a formula II: SLC site - polypeptide II or a capture molecule of formula III: SLC site - internal complementary sequence III wherein the SLC site comprises from about 17 to about 22 bases in length and is recognized by a replicase enzyme; wherein the linker moiety comprises an organic linking moiety having from about two to about ten carbon atoms in length (straight or branched); wherein HO« denotes an active hydroxyl moiety that is able to bind to the 5'-m⁷G(5')ppp(5')G cap structure of an RNA molecule with high affinity; and wherein an internal complementary sequence is from about 10 to about 50 bases in length and complementary to a region of the RNA molecule to be amplified.

Preferably, the process further comprises (c) preventing the amplified RNA molecules from being degraded. Preferably, the capture molecule is a cap-capture molecule. Preferably, the capture molecule containing replicase recognition SLC site sequence is a sequence selected from the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 and combinations thereof. Preferably, the linker moiety of the cap-capture molecule is selected from the group consisting of branched and straight -Ci-β alkyl-, -Cι.₆ alkenyl-, and -Cι-6 alkoxy-. Preferably, the rNTPs are present in an approximately equimolar concentration. Preferably, the cap- capture molecule is a polypeptide having a high affinity to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule. Most preferably, the polypeptide is an antibody against the cap structure. Preferably, the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7, T3 and SP6 RNA polymerase, Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

Preferably, the inventive process further comprises a first step of capping the 5' end of

■ 1 " the source RNA. Most preferably, the capping reagent is by P -5'-(7-methyl)-guanosιne-P -5'- adenosine-triphosphate or P¹-5'-(7-methyl)-guanosine-P³-5'-guanosine-triphosphate. Preferably, the source RNA material is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA transcription-based amplified RNA (such as TTR-amplified RNA or other DNA-dependent RNA polymerase transcribed RNA), RNA-promoter-driven transcribed RNA, aRNA, aRNA-amplified RNA, single-cell mRNA library, isolated mRNA, RNA contained within cells, and combinations of RNA sources. Preferably, the source RNA is prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration. Most preferably, the fixed cells are obtained from fixative-treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin-embedded tissues on slides.

The present invention further provides a replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures, comprising: a. contacting an RNA source with a plurality of cap-capture molecules coupled to a replicase recognition site (SLC) to form a cap-captured RNA source, wherein the cap-capture molecules comprise moieties capable of binding to the 5 '-cap structures of said messenger RNAs; and b. incubating the cap-captured RNA source with a solution comprising (1) a capture molecule, (2) a plurality of ribonucleotides (rNTPs), and (3) a replicase or RNA- dependent RNA polymerase enzyme activity, wherein the capture molecule has a formula I: SLC site - linker moiety - HO» I or a cap-capture molecule having a formula II: SLC site - polypeptide II or a capture molecule of formula III

SLC site - internal complementary sequence III wherein the SLC site comprises from about 17 to about 22 bases in length and is recognized by a replicase enzyme; wherein the linker moiety comprises an organic linking moiety having from about two to about ten carbon atoms in length (straight or branched); wherein HO» denotes an active hydroxyl moiety that is able to bind to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule with high affinity; and wherein an internal complementary sequence is from about 10 to about 50 bases in length and complementary to a region of the RNA molecule to be amplified, whereby template extension of the replicase recognition site forms a plurality of replicated copies of the source RNAs. Preferably, the process further comprises (c) preventing the amplified RNA molecules from being degraded. Preferably, the capture molecule is a cap-capture molecule. Preferably, the capture molecule containing a replicase recognition SLC site sequence is a sequence selected from the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 and combinations thereof. Preferably, the linker moiety of the cap-capture molecule is selected from the group consisting of branched and straight -Cι-₆ alkyl-, -Cι_-6 alkenyl-, and -Cι-6 alkoxy-. Preferably, the cap-capture molecule is a polypeptide having a high affinity to the 5'- m⁷G(5')ppp(5')G cap structure of an mRNA molecule. Most preferably, the polypeptide is an antibody against the cap structure. Preferably, the rNTPs are present in an approximately equimolar concentration. Preferably, the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7, T3 SP6 RNA polymerase, Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

Preferably, the inventive process further comprises a first step of capping the 5' end of the source RNA. Most preferably, the capping reagent is by P¹-5'-(7-methyl)-guanosine-P³-5'- adenosine-triphosphate or P¹-5'-(7-methyl)-guanosine-P³-5'-guanosine-triphosphate. Preferably, the source RNA material is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA transcription-based Amplified RNA such as TTR-amplified RNA or other DNA-dependent RNA polymerase transcribed RNA, RNA- promoter-driven transcribed RNA, aRNA, aRNA-amplified RNA, single cell RNA library, isolated mRNA, RNA contained within cells, and combinations of RNA sources. Preferably, the source RNA is prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration. Most preferably, the fixed cells are obtained from fixative-treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin-embedded tissues on slides. The present invention further provides a process for a replicase or RNA-dependent RNA polymerase cycling process coupled with a DNA-dependent RNA polymerase-driven RNA transcription reaction (tandem transcription reaction (TTR)) for amplifying an RNA source, comprising: (a) contacting source messenger RNAs with a plurality of oligodeoxythymidylate primers coupled to a replicase recognition site and a RNA promoter, wherein the oligodeoxythymidylate primers are capable of binding to the 3 '-end poly(A) tail structures of said messenger RNAs; wherein the order of the;

(b) primer extending the 5'-RNA promoter sequence-replicase recognition SLC site-oligodeoxythymidylate-3 ' primer to form a plurality of complementary DNA copies of the source messenger RNAs, wherein the complementary DNAs are formed by reverse transcription activity; (c) amplifying the complementary DNAs from the RNA promoter to form a plurality of transcript copies of antisense RNAs having the replicase recognition SLC site sequence; and

(d) amplifying the antisense RNAs with replicase activity recognizing the replicase recognition SLC site to form a plurality of replicated copies of the antisense RNAs.

Preferably, the order of elements in the primer is 5' -RNA promoter sequence-replicase recognition SLC site-oligodeoxythymidylate-3'. Preferably, the process further comprises double stranding the cDNA to form double-stranded DNAs.

The present invention further provides a kit for amplifying source RNA to use with gene expression assays, comprising (1) a capture molecule, (2) a plurality of ribonucleotides

(rNTPs), and (3) a replicase or RNA-dependent RNA polymerase enzyme activity, wherein the capture molecule is a cap-capture molecule having a formula I: SLC site - linker moiety - HO« I or a cap-capture molecule having a formula II: SLC site - polypeptide II or a capture molecule of formula III: SLC site - internal complementary sequence III wherein the SLC site comprises from about 17 to about 22 bases in length and is recognized by a replicase enzyme; wherein the linker moiety comprises an organic linking moiety having from about two to about ten carbon atoms in length (straight or branched); wherein HO« denotes an active hydroxyl moiety that is able to bind to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule with high affinity; and wherein an internal complementary sequence is from about 10 to about 50 bases in length and complementary to a region of the RNA molecule to be amplified. Preferably, the capture molecule is a cap-capture molecule. Preferably, the replicase recognition SLC site sequence is a sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and combinations thereof. Preferably, the linker moiety of the cap-capture molecule is selected from the group consisting of branched and straight -Cι-₆ alkyl-, -Cι-₆ alkenyl-, and -Cι_-6 alkoxy-. Preferably, the cap-capture molecule is a polypeptide having a high affinity to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule. Most preferably, the said polypeptide is an antibody against the cap structure.

Preferably, the rNTPs are present in an approximately equimolar concentration. Preferably, the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7, T3 and SP6RNA polymerase, Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

Preferably, the inventive process further comprises a first step of capping the 5 ' end of the source RNA. Most preferably, the capping reagent is by P¹-5'-(7-methyl)-guanosine-P³-5'- adenosine-triphosphate or P ' -5 ' -(7-methyl)-guanosine-P³-5 ' -guanosine-triphosphate. Preferably, the source RNA material is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA transcription-based amplified RNA, TTR-amplified RNA, DNA-dependent RNA polymerase transcribed RNA, RNA-promoter- driven transcribed RNA, aRNA, aRNA-amplified RNA, single cell RNA library, isolated mRNA, RNA contained within cells, and combinations of RNA sources. Preferably, the source RNA is prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration. Most preferably, the fixed cells are obtained from fixative-treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin-embedded tissues on slides. Brief Description of the Drawings Figure 1 shows a schematic of the inventive process using a "cap" capture molecule and an mRNA source to amplify into cRNA.

Figures 2a and 2b show agarose gels of the experiments conducted in example 2 using a cap-capture molecule.

Figure 3 shows a schematic of the inventive process using a capture molecule containing a complementary sequence to target RNA.

Figure 4 shows the data on a 1% formaldehyde-agarose gel electrophoresis from a one- step amplification process of Example 3 that increased the amount of original RNA template to about 100 fold.

Figure 5 shows a schematic of combining the inventive RCR process with the inventive TTR process to amplify source RNA to an even greater extent.

Figure 6 shows an alternative scheme of the inventive TTR process to amplify source RNA to an even greater extent. Detailed Description of the Invention

Although specific embodiments of the present invention will now be described with reference to the drawings, it should be understood that such embodiments are by way of example only and merely illustrative of but a small number of the many possible specific embodiments which can represent applications of the principles of the present invention. Various changes and modifications obvious to one skilled in the art to which the present invention pertains are deemed to be within the spirit, scope and contemplation of the present invention. Definitions

"Antisense RNA" (aRNA or cRNA) refers to an RNA sequence that is complementary to a natural messenger RNA sequence in an A-U and C-G composition.

"Antisense sequence" refers to a nucleotide sequence that is complementary to its respective mRNA homologue.

"Cap Capture Molecule" is a chemical or a protein such as an antibody that is able to bind to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule with high affinity.

"Capture Molecule" refers to either a cap-capture molecule having a formula SLC site - linker - HO«, or SLC site - complimentary sequence. The capture molecule serves to both bind to the RNA molecule at the 5' end (in the case of a cap-capture molecule) or internally and provide a recognition template for the appropriate replicase enzyme.

"Complementary DNA" (cDNA) refers to a DNA sequence that is complementary to a natural messenger RNA sequence in an A-T and C-G composition. "Oligo(dT)-promoter sequence" refers to an RNA polymerase promoter sequence coupled with a poly-deoxythymidylate (dT)n sequence in its 3 '-end, of which the minimal number of linked dT is seven; most preferably, the number is about twenty-six.

"Ribonucleotide" or rNTP is a collection of ATP, GTP, CTP and UTP, preferably at about equimolar amounts. "Replicase" or RNA-dependent RNA polymerase refers to an enzymatic activity.

Many replicases are RNA-dependent RNA polymerases (Khoshnan et al., J. Virol. 68: 7108- 7114 (1994), except some DNA-dependent RNA polymerases such as viral polymerases. Particularly useful replicases include, for example, Brome mosaic virus (BMV) replicase (Kim et al., RNA 7:1476-1485 (2001)), Togaviridae virus replicase (Boorsma et al., Nature Biotech. 18:429-432 (2000)), Flock house virus (FHV) replicase (Johnson et al., J Virol. 71 :3323-3327 (1997)), Q beta replicase (Mills, J. Mol. Biol. 200:489-500 (1988)) and the like.

"Sense sequence" refers to a nucleotide sequence that is in the same sequence order and composition as its homolog mRNA.

"Source RNA Material" refers to any RNA material obtained from or isolated from any source, for example, single cells, cultured cells, tissues, RNA transcription-based amplified RNA such as TTR-amplified RNA or other DNA-dependent RNA polymerase transcribed RNA, RNA-promoter-driven transcribed RNA, aRNA and aRNA-amplified RNA, single cell RNA library, isolated mRNA, RNA contained within cells, and combinations of RNA sources. "SLC Recognition Site" or stem-loop-cytosine (SLC) site is introduced into the ends of or within a messenger RNA sequence. An SLC site is generally from about 17 to about 22 bases in length and is recognized by a replicase enzyme. An example of an SLC site sequence is 5'-GAAUGGGCCC CAUAAUG-3' [SEQ ID NO. 1] recognized by a BMV replicase.

The present invention provides a RNA replication reaction method using replicases and capture molecules for mRNA (preferably eukaryotic) amplification. This process is called "replicase cycling reaction" (RCR). RCR is particularly well-suited for differential screening of tissue-specific gene expressions at single cell level, cloning full-length sequences of unknown gene transcripts, generating pure probes for hybridization assays, synthesizing peptides in vitro, and preparing complete antisense mRNA libraries for microarray technologies. The essential element of RCR is cycling steps of either a 5'-cap/phosphate- induced extension of mRNA templates by RNA-dependent replicase activity or a complementary sequence moiety capture molecule having a SLC recognition site to extend an mRNA template by an RNA-dependent replicase activity. In brief, the preferred version (Figure 1) using a cap-capture molecule provides: a) prevention of mRNA degradation, b) 5'- cap or 5 '-phosphate initiation of replicase activity, c) RNA sequence extension, and d) repeating aforementioned steps to achieve desired RNA amplification. The advantage is that the steps can all be done with many cycles in a single incubation over relatively short time.

The mRNAs can be prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration. Those fixed cells include fixative-treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin-embedded tissues on slides. In another aspect of this embodiment, said amplified messenger RNAs are preferably capped by P^l-5'-(7-methyl)- guanosine-P -5'-adenosine-triphosphate or P -5'-(7-methyl)-guanosine-P -5'-guanosine- triphosphate in the step (c) for further in vitro translation. The replicase recognition site can be either a stem-loop-cytosine (SLC) RNA motif or a RNA promoter (RP) domain which contains high binding affinity to viral replicases selected from but not limited by Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase and Q beta replicase. Preferably, the resulting amplified RNAs can be subsequently reverse-transcribed and double-stranded into double-stranded cDNA for cloning into competent vectors.

The principle of the preferred embodiment relating to the tandem transcription reaction or TTR relies on repeating steps of reverse transcription, double-stranded cDNA synthesis, in vitro transcription and then cRNA or sense RNA replication to amplify desired RNA sequences up to two million folds in one cycle. In brief, the preferred version of the present invention is based on: a) denaturation of starting nucleic acid templates, b) reverse transcription and or DNA polymerization to incorporate an RNA promoter and a replicase recognition site such as an SLC site sequence into the 5 '-end of complementary templates or even internally within an RNA template, c) transcription of cRNAs from the DNA-dependent RNA promoter, d), replication of the cRNAs from the replicase recognition site, and e) repeating the aforementioned steps (a)-(d) to achieve the desired RNA amplification. Cap-Capture Molecule

The present invention utilizes capture molecules, and in particular cap-capture molecules, that bind to a replicase recognition site or stem-loop-cytosine (SLC) site. The SLC site is introduced into the ends of or within a messenger RNA sequence. Following template replacement between the SLC and mRNA sequence, the present inventive process provides for amplification of more than one thousand mRNA copies by only one unit of replicase activity. The conjugation of a SLC site (such as 5'-GAAUGGGCCC CAUAAUG-3' [SEQ ID NO. 1]) to the cap-capture molecule is completed during oligonucleotide synthesis. Based on phosphoric acid alkylation (Solomons, "Organic Chemistry, 6th Edition", pp437 & 720 (1996)), the cap capture reaction can be induced in the presence of dehydrate reagents, such as formaldehyde or acetaldehyde. The addition of formaldehyde, an RNase inhibitor, actually further protects RNAs from degradation. To remove formaldehyde during RNA replication, a pre-incubation at 65 °C for 10 min must be completed before enzymatic reactions. Such pre- incubation also minimizes the secondary structures of RNAs for better full-length product formation. The cap-capture molecule can be also a polypeptide such as an antibody having a strong affinity to the cap structure. The conjugation of the oligonucleotide SLC sequence onto an antibody for the cap structure can be performed by skilled in the art.

The replicase cycling reaction is preferably performed in buffered conditions similar to in vitro transcription. For example, buffered conditions such as those used in Example 2 are preferred. Another example of a buffered condition is in lx RT&T buffer: 50mM Tris-HCl, pH 8.2 at 25 °C, 40mM KC1, 8mM MgCl₂, lOmM DTT, 5μg/ml BSA. Stable and efficient reaction conditions occurs during the first two-hour incubation at about 30~35°C. The rate of RNA synthesis decreases (40-50%) after three-hour incubation or below 30 °C incubation. Longer reactions may increase yield, but the possibility of degradation by RNase increases. Occasionally gentle mixing can prevent the stall of crowded RNA polymerization on a template and enhance full-length synthesis. The overall rate of RNA synthesis is maximal between pH 7.7 and 8.3, but it remains about 70% of maximum at pH 7.0 or 9.0. High concentrations of NaCl, KC1 or NH C1 above 75 mM will inhibit the reaction and should be avoided.

Internal Replicase Recognition Site Sequence (Internal Capture Molecule) Generated by TTR

The present invention further provides an amplification of nucleic acid sequence process utilizing a tandem transcription reaction (TTR) as the name for the inventive process using an internal replicase recognition site sequence or internal capture molecule artificially introduced into the source RNA by the TTR process. In one preferred embodiment of a tandem transcription reaction (TTR), a double strand polynucleotide sequence contains a promoter sequence of a DNA-dependent RNA polymerase followed by a template sequence or replication initiation site such as the SLC site sequence for the replicase or RNA-directed RNA polymerase. The polynucleotide sequence is first to be transcribed into hundreds to thousands copies of RNA transcripts by the DNA-dependent RNA polymerase. The resultant transcripts contain at their 5 '-end the template sequence or the initiation site for the RNA-directed RNA polymerase or the SLC site for the replicase. Upon transcription by the replicase or the RNA- directed RNA polymerase, the polynucleotide sequences are to be copied by about another few thousand to million folds. Hence, the original polynucleotide sequence can be amplified by hundreds of thousands folds to potentially a billion folds of RNA transcripts of the desired target DNA or RNA of interest.

In another embodiment of the inventive process for amplifying a specific DNA sequence, a specific DNA sequence is hybridized with an anti-sense oligonucleotide containing the sequences for the RNA- and DNA-dependent RNA polymerases and polymerization reaction is carried out to make double stranded (ds) copy of the original sequence. The TTR is performed first with DNA-dependent RNA polymerase followed by replicase or RNA- dependent RNA polymerase generating hundreds thousands to billions copies of RNA transcripts of same or opposite orientation of the original sequence. In another embodiment of the inventive process for amplifying a specific RNA sequence such as an mRNA transcript, the mRNA transcript sequence is reversed-transcribed into complementary DNA (cDNA) sequence by a reverse transcriptase with a similar primer as describe in the above embodiment. The cDNA sequence is further made into ds cDNA and resultant ds cDNA sequence is functioned as template for the TTR amplification as described in the above embodiment.

In yet another embodiment of the inventive process, a total pool of mRNA transcripts from a cell or tissue source are reverse transcribed into cDNA by an oligo dT primer tagged with the TTR sequence containing a RNA-dependent RNA polymerase recognition site or the SLC site sequence and a DNA-dependent RNA promoter at its 5 '-end. The cDNAs are then made into ds cDNA with methods known in the art, such as RNase H-mediated RNA degradation and double stranding or with specific gene(s)-primer extension. The resultant ds cDNAs are used as templates for the TTR amplification process. The mRNA transcripts are copied 10⁵ to 10⁹ fold into anti-sense RNA transcripts. In yet another embodiment, the mRNA transcripts are reverse transcribed into cDNAs, the 3 '-end of the cDNAs are to be tailed by terminal transferase (TdT) of multiple nucleotides, such as poly (dG)_n tail. The poly (dG)_n tail is hybridized to a poly (dC)_n oligonucleotide primer tagged with the TTR sequence containing a RNA-dependent RNA polymerase recognition site or the SLC site sequence and a DNA-dependent RNA promoter at its 5 '-end, ds cDNAs are made and the resultant ds cDNAs are used as templates for the TTR amplification.

In yet another embodiment of the inventive process, genomic DNAs as a source is amplified by the inventive TTR process. The genomic DNA is to be digested with certain restriction enzyme or shear force to pieces of fragmented DNA sequences of hundreds to thousands base pairs (bps) in length. The resultant DNA sequences are ligated with ds linker sequence tagged with TTR sequence containing a RNA-dependent RNA polymerase recognition site or SLC site sequence and a DNA-dependent RNA promoter, and used as templates for the TTR amplification. The whole genome sequences of the genomic DNAs can be copied, though in pieces, into hundreds thousands to billions of copies. The resultant RNA transcripts of TTR amplification, either in the same or opposite orientation of the original nucleotide sequences, are converted into single-strand or ds DNA sequences by methods known in the art, such as RT-PCR, for further cloning and gene expression and genetic analysis.

The tandem transcription reaction (TTR) process is useful for linear amplification of specific nucleic acid sequence or nucleic acid sequences such as mRNAs or whole genome sequences. The TTR reaction process can be repeated or cycled more than once if needed. Applications of the Inventive Process

The inventive process can be used to prepare labeled antisense RNA (cRNA) probes for microarray analysis. The final nucleotide products (i.e., amplified RNA) can be labeled in the form of said antisense messenger RNAs for microarray analysis. Although certain preferred embodiments of the present invention have been described, the spirit and scope of the invention is by no means restricted to what is described above. For example, within the general framework of: a) one or more replicase-recognition-site-containing primers; b) one or more RNA promoter-containing primers; c) one or more species of starting messenger RNAs; d) one or more kinds of replicases used in one reaction; and e) one or more rounds of the cycling steps for RNA amplification, there is a very large number of permutations and combinations possible, all of which are within the scope of the present invention.

EXAMPLE 1 Cell Fixation and Permeabilisation MCF7 cells, a breast cancer cell line, were grown in DMEM medium supplemented with 10% fetal calf serum. One 70% confluent 60mm dish culture was trypsinized, collected and washed three times in 5ml phosphate buffered saline (PBS, pH 7.2) at room temperature, then suspended in 1ml of ice-cold 10% formaldehyde solution in 0.15M NaCl. After one hour incubation on ice with occasional agitation, the cells were centrifuged at 13,000 rpm for 2 min and wash three times in ice-cold PBS with vigorous pipetting. The collected cells were resuspended in 0.5% non-ionic detergents, such as (octylphenoxy)-polyethanol or polyoyethylenesorbitan and incubated for one hour with frequent agitation. After that, three washes were given to cells in ice-cold PBS containing 0.1M glycine and the cells were resuspended in 1ml of the same buffer with vigorous pipetting in order to be evenly separated into small aliquots and stored at -70 C for up to a month.

EXAMPLE 2 Replicase Cycling Reaction

For the in vitro replication of mRNAs in cells, one hundred of the fixed cells (from example 1 herein) were thawed, resuspended in 20μl of DEPC-treated ddH₂O, mixed with 25pmol stem-loop-containing cap-capture molecules (HO«-5'-GAAUGGGCCC CAUAAUG- 3', SEQ ID NO.l), heated to 65 C for 10 min and then cooled on ice. The functional group of the cap-capture molecules was indicated by HO« symbol, showing its chemically active affinity to the 5'- phosphate of m⁷G(5')ppp(5')G cap structure of mRNA molecules. The cap capture reaction occurs based on the high incidence rate of phosphoric acid alkylation under dehydration conditions. A 50μl reaction was prepared, comprising 5μl of 1 Ox RT&T buffer

(500mM Tris-HCl, pH 8.2 at 25°C, 400mM KC1, 80mM MgCl₂, lOOmM DTT, 50μg/ml BSA), rNTPs (ImM each for ATP, GTP, CTP and UTP, and 0.2mM P¹-5'-(7-methyl)-guanosine-P³- 5'-guanosine-triphosphate), RNase inhibitor (10U) and the above cells. After BMV replicase (50U) was added, the reaction was mixed and incubated at 35°C for 60 min. This procedure amplified the original mRNAs up to 1,000 fold (Figure 1 and Figure 5, steps (a) to (c)).

As shown in Figure 5, steps (a') to (e'), the amplified mRNAs were purified by a microcon-50 filter and applied to a reverse transcription reaction (20μl) on ice, comprising 2 μl of lOx RT&T buffer, 1 μM stem-loop-containing and RNA polymerase promoter-linked poly (dT) primer (dephosphated 5'-dCCAGTGAATT GTAATACGAC TCACTATAGG GAAUGGGCCC CAUATTTTTT TTTTTT-3', [SEQ ID NO.2]), dNTPs (ImM each for dATP, dGTP, dCTP and TTP) and RNase inhibitors (10U). After MMLV reverse transcriptase (20U) and Pwo DNA polymerase (5U) was added, the reaction was incubated at 35 °C for 60 min and then at 68 °C for 10 min. The cDNAs so obtained were collected in an in vitro transcription reaction (50 μl), comprising 3 μl of lOx RT&T buffer and rNTPs (ImM each for ATP, GTP, CTP and UTP). After the mixture of T7 RNA polymerase (2,000U) and BMV replicase (50U) was added, the reaction was incubated at 35 °C for 60 minutes. After two cycles of replicase and transcription amplification, the quality of RNA products can be assessed on a 1% formaldehyde-agarose gel as shown in Figure 2a. The over-expression of β- catenin, a breast cancer marker, can be detected clearly in MCF7 breast cancer cells but not LNCaP prostate cancer cells by Northern blot analysis as shown in Figure 2b.

EXAMPLE 3 Sequence-Specific Replicase Cycling Reaction For the in vitro replication of specific RNA species, about lng of starting bcl-2 RNA template was mixed with about 25pmol SLC-containing bcl-2-specific complementary primers (5'-CTTCTTCAGG CCAGGGAGGC GAAUGGGCCC CAUAAUG-3', [SEQ ID NO.3]) in 20 μl of DEPC-treated ddH₂O, heated to 65 C for 10 min and then cooled on ice. A 50μl reaction was prepared, comprising 5μl of lOx RT&T buffer (500mM Tris-HCl, pH 8.2 at 25 °C, 400mM KCl, 80 mM MgCl₂, 100 mM DTT, 50 μg/ml BSA), rNTPs (ImM each for ATP, GTP, CTP and UTP, and 0.2mM P¹-5'-(7-methyl)-guanosine-P³-5'-guanosine-triphosphate), RNase inhibitor (10U) and the above mixture. After BMV replicase (50U) was added, the reaction was mixed and incubated at 35 C for 60 min. This one-step amplification increased the amount of original RNA template to about 100 fold based on a 1% formaldehyde-agarose gel electrophoresis as shown in Figure 4.

SEQUENCE LISTING (1) GENERAL INFORMATION :

(iii) NUMBER OF SEQUENCES: 3

(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic"

(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GAAUGGGCCC CAUAAUG 17

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic" (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

CCAGTGAATT GTAATACGAC TCACTATAGG GAAUGGGCCC CAUATTTTTT TTTTTT 56

(2) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic" (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

CTTCTTCAGG CCAGGGAGGC GAAUGGGCCC CAUAAUG 37

Claims

We claim:

1. A replicase cycling process for amplifying an RNA source, comprising:

(a) providing a source RNA material for amplification; and

(b) incubating the source RNA material with a solution comprising (1) a capture molecule, (2) a plurality of ribonucleotides (rNTPs), and (3) a replicase or RNA-dependent

RNA polymerase enzyme activity, wherein the capture molecule has a formula I:

SLC site - linker moiety - HO« I or a cap-capture molecule having a formula II:

SLC site - polypeptide II or a capture molecule of formula III:

SLC site - internal complementary sequence III wherein the SLC site comprises from about 17 to about 22 bases in length and is recognized by a replicase enzyme; wherein the linker moiety comprises an organic linking moiety having from about two to about ten carbon atoms in length (straight or branched); wherein HO» denotes an active hydroxyl moiety that is able to bind to the 5'-m⁷G(5')ppp(5')G cap structure of an RNA molecule with high affinity; and wherein an internal complementary sequence is from about 10 to about 50 bases in length and complementary to a region of the RNA molecule to be amplified.

2. The replicase cycling process for amplifying an RNA source of claim 1 wherein the process further comprises (c) preventing the amplified RNA molecules from being degraded.

3. The replicase cycling process for amplifying an RNA source of claim 1 wherein the capture molecule is a cap-capture molecule.

4. The replicase cycling process for amplifying an RNA source of claim 1 wherein the capture molecule containing replicase recognition SLC site sequence is a sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and combinations thereof.

5. The replicase cycling process for amplifying an RNA source of claim 1 wherein the linker moiety of the cap-capture molecule is selected from the group consisting of branched and straight -Cι-6 alkyl-, -Cι-6 alkenyl-, and -Cι-₆ alkoxy-.

6. The replicase cycling process for amplifying an RNA source of claim 1 wherein the rNTPs are present in an approximately equimolar concentration.

7. The replicase cycling process for amplifying an RNA source of claim 1 wherein the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7, T3 and SP6 RNA polymerase, Brome mosaic virus (BMV) replicase,

Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

8. The replicase cycling process for amplifying an RNA source of claim 1 wherein the cap-capture molecule is a polypeptide having a high affinity to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule.

9. The replicase cycling process for amplifying an RNA source of claim 8 wherein the polypeptide is an antibody against the cap structure.

10. The replicase cycling process for amplifying an RNA source of claim 1 wherein the inventive process further comprises a first step of capping the 5' end of the source RNA.

11. The replicase cycling process for amplifying an RNA source of claim 10 wherein the capping reagent is by P¹-5'-(7-methyl)-guanosine-P³-5'-adenosine-triphosphate or P'-5 '-(7-methyl)-guanosine-P³-5 '-guanosine-triphosphate.

12. The replicase cycling process for amplifying an RNA source of claim 1 wherein the source RNA material is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA transcription-based amplified RNA (such as TTR-amplified RNA or other DNA-dependent RNA polymerase transcribed RNA), RNA-promoter-driven transcribed RNA, aRNA, aRNA-amplified RNA, single-cell mRNA library, isolated mRNA, RNA contained within cells, and combinations of RNA sources.

13. The replicase cycling process for amplifying an RNA source of claim 12 wherein the source RNA is prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration.

14. The replicase cycling process for amplifying an RNA source of claim 13 wherein the fixed cells are obtained from laser-capture-machine-dissected cells, fixative- treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin-embedded tissues on slides.

15. A replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures, comprising: a. contacting an RNA source with a plurality of cap-capture molecules coupled to a replicase recognition site (SLC) to form a cap-captured RNA source, wherein the cap-capture molecules comprise moieties capable of binding to the 5 '-cap structures of said messenger RNAs; and b. incubating the cap-captured RNA source with a solution comprising (1) a capture molecule, (2) a plurality of ribonucleotides (rNTPs), and (3) a replicase or RNA- dependent RNA polymerase enzyme activity, wherein the capture molecule has a formula I: SLC site - linker moiety - HO» I or a cap-capture molecule having a formula II:

SLC site - polypeptide II or a capture molecule of formula III SLC site - internal complementary sequence III wherein the SLC site comprises from about 17 to about 22 bases in length and is recognized by a replicase enzyme; wherein the linker moiety comprises an organic linking moiety having from about two to about ten carbon atoms in length (straight or branched); wherein HO« denotes an active hydroxyl moiety that is able to bind to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule with high affinity; and wherein an internal complementary sequence is from about 10 to about 50 bases in length and complementary to a region of the RNA molecule to be amplified, whereby template extension of the replicase recognition site forms a plurality of replicated copies of the source RNAs.

16. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5'-cap structures of claim 15, wherein the process further comprises (c) preventing the amplified RNA molecules from being degraded.

17. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the capture molecule containing a replicase recognition SLC site sequence is a sequence selected from the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 and combinations thereof.

18. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the linker moiety of the cap- capture molecule is selected from the group consisting of branched and straight -Cι-₆ alkyl-, - Cι_-6 alkenyl-, and -Cι_₆ alkoxy-.

19. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the cap-capture molecule is a polypeptide having a high affinity to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule.

20. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 19, wherein the polypeptide is an antibody against the cap structure.

21. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the rNTPs are present in an approximately equimolar concentration.

22. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7, T3 SP6 RNA polymerase, Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

23. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the inventive process further comprises a first step of capping the 5' end of the source RNA.

24. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5'-cap structures of claim 15, wherein the capping reagent is by P'-5'- (7-methyl)-guanosine-P³-5'-adenosine-triphosphate or P¹-5'-(7-methyl)-guanosine-P³-5'- guanosine-triphosphate.

25. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the source RNA material is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA transcription-based Amplified RNA such as TTR-amplified RNA or other DNA- dependent RNA polymerase transcribed RNA, RNA-promoter-driven transcribed RNA, aRNA, aRNA-amplified RNA, single cell RNA library, isolated mRNA, RNA contained within cells, and combinations of RNA sources.

26. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 15, wherein the source RNA is prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration.

27. The replicase cycling reaction (RCR) process for amplifying either sense or antisense RNAs having 5 '-cap structures of claim 26, wherein the fixed cells are obtained from fixative-treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin- embedded tissues on slides.

28. A process for a replicase or RNA-dependent RNA polymerase cycling process coupled with a DNA-dependent RNA polymerase-driven RNA transcription reaction (tandem transcription reaction (TTR)) for amplifying an RNA source, comprising:

(a) contacting source messenger RNAs with a plurality of oligodeoxythymidylate primers coupled to a replicase recognition site and a RNA promoter, wherein the oligodeoxythymidylate primers are capable of binding to the 3 '-end poly(A) tail structures of said messenger RNAs; wherein the order of the; (b) primer extending the 5' -RNA promoter sequence-replicase recognition SLC site-oligodeoxythymidylate-3' primer to form a plurality of complementary DNA copies of the source messenger RNAs, wherein the complementary DNAs are formed by reverse transcription activity;

(c) amplifying the complementary DNAs from the RNA promoter to form a plurality of transcript copies of antisense RNAs having the replicase recognition SLC site sequence; and

29. The process for a replicase or RNA-dependent RNA polymerase cycling process coupled with a DNA-dependent RNA polymerase-driven RNA transcription reaction (tandem transcription reaction (TTR)) for amplifying an RNA source, wherein the order of elements in the primer is 5' -RNA promoter sequence-replicase recognition SLC site- oligodeoxythymidylate-3 ' .

30. The process for a replicase or RNA-dependent RNA polymerase cycling process coupled with a DNA-dependent RNA polymerase-driven RNA transcription reaction (tandem transcription reaction (TTR)) for amplifying an RNA source, wherein the process further comprises double stranding the cDNA to form double-stranded DNAs.

31. A kit for amplifying source RNA to use with gene expression assays, comprising (1) a capture molecule, (2) a plurality of ribonucleotides (rNTPs), and (3) a replicase or RNA-dependent RNA polymerase enzyme activity, wherein the capture molecule is a cap-capture molecule having a formula I: SLC site - linker moiety - HO« I or a cap-capture molecule having a formula II:

SLC site - polypeptide II or a capture molecule of formula III: SLC site - internal complementary sequence III wherein the SLC site comprises from about 17 to about 22 bases in length and is recognized by a replicase enzyme; wherein the linker moiety comprises an organic linking moiety having from about two to about ten carbon atoms in length (straight or branched); wherein HO» denotes an active hydroxyl moiety that is able to bind to the 5'-m⁷G(5')ppp(5')G cap structure of an mRNA molecule with high affinity; and wherein an internal complementary sequence is from about 10 to about 50 bases in length and complementary to a region of the RNA molecule to be amplified.

32. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the cap-capture molecule is a polypeptide having a high affinity to the 5'- m⁷G(5')ppp(5')G cap structure of an mRNA molecule.

33. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the polypeptide is an antibody against the cap structure.

34. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the rNTPs are present in an approximately equimolar concentration.

35. The kit for amplifying source RNA to use with gene expression assays of claim 34 wherein the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7, T3 and SP6RNA polymerase, Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

36. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the inventive process further comprises a first step of capping the 5 ' end of the source RNA.

37. The kit for amplifying source RNA to use with gene expression assays of claim 36 wherein the capping reagent is by P¹-5'-(7-methyl)-guanosine-P³-5'-adenosine-triphosphate or P -5'-(7-methyl)-guanosine-P -5'-guanosine-triphosphate.

38. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the source RNA material is obtained from a source selected from the group consisting of single cells, cultured cells, tissues, RNA transcription-based amplified RNA, TTR-amplified RNA, DNA-dependent RNA polymerase transcribed RNA, RNA-promoter- driven transcribed RNA, aRNA, aRNA-amplified RNA, single cell RNA library, isolated mRNA, RNA contained within cells, and combinations of RNA sources.

39. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the source RNA is prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration.

40. The kit for amplifying source RNA to use with gene expression assays of claim 39 wherein the fixed cells are obtained from fixative-treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin-embedded tissues on slides.

41. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the replicase recognition SLC site sequence is a sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and combinations thereof.

42. The kit for amplifying source RNA to use with gene expression assays of claim 31 wherein the linker moiety of the cap-capture molecule is selected from the group consisting of branched and straight -Cι_6 alkyl-, -Cι-₆ alkenyl-, and -Cι-6 alkoxy-.

43. A replicase cycling reaction process for amplifying an RNA source, comprising:

(a) providing a source RNA material containing, within the RNA molecule, artificially introduced recognition site sequence such as SLC site sequence for replicase or RNA-dependent RNA polymerase; and

(b) incubating the source RNA material with a solution comprising (1) a plurality of ribonucleotides (rNTPs) and (2) a replicase or RNA-dependent RNA polymerase enzyme activity, wherein the SLC site comprises from about 17 to about 35 bases in length and is recognized by a replicase enzyme.

44. The process for replicase cycling reaction process for amplifying an RNA source of claim 43 wherein the introduction of the recognition site sequence such as SLC site for replicase or RNA-dependent RNA polymerase into source RNA is by an RNA transcription-based RNA amplification method.

45. The process for replicase cycling reaction process for amplifying an RNA source of claim 43 wherein the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7, T3 and SP6 RNA polymerase, Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

46. The process for replicase cycling reaction process for amplifying an RNA source of claim 43 wherein the source RNA material is obtained from a source selected from the group comprising RNA transcription-based amplified RNA such as TTR-amplified RNA or other DNA-dependent RNA polymerase transcribed RNA, RNA-promoter-driven transcribed RNA, aRNA and aRNA-amplified RNA, single cell RNA library, and combinations of RNA sources.

47. A kit for amplifying source RNA material containing, within the RNA molecule, artificially introduced recognition site sequence such as SLC site sequence for replicase or RNA-dependent RNA polymerase, comprising (1) a plurality of ribonucleotides (rNTPs) and (2) a replicase or RNA-dependent RNA polymerase enzyme activity, wherein the SLC site comprises from about 17 to about 35 bases in length and is recognized by a replicase enzyme

48. The kit for amplifying source RNA to use with gene expression assays of claim 47 wherein the process further comprises (c) preventing the amplified RNA molecules from being degraded.

49. The kit for amplifying source RNA to use with gene expression assays of claim 47 wherein the SLC site sequence is a sequence comprising SEQ ID NO. 1.

50. The kit for amplifying source RNA to use with gene expression assays of claim 47 wherein the replicase or RNA-dependent RNA polymerase enzyme activity is selected from the group consisting of T7 replicase, T3 and SP6 RNA polymerase, Brome mosaic virus (BMV) replicase, Trichomonas vaginalis virus (TW) replicase, Flock house virus (FHV) replicase, Q beta replicase, and combinations thereof.

51. The kit for amplifying source RNA to use with gene expression assays of claim 47 wherein the source RNA material is obtained from a source selected from the group comprising RNA transcription-based amplified RNA such as TTR-amplified RNA or other DNA-dependent RNA polymerase transcribed RNA, RNA-promoter-driven transcribed RNA, aRNA and aRNA-amplified RNA, single cell RNA library, and combinations of RNA sources.