RNA amplification

Amplification of nucleic acids also include RNA amplifications. For instance, the heat-shock mRNA (103-mer) in viable Cryptosporidium parvum in water treatment plants was detected after amplification by nucleic acid sequence-based amplification (NASBA). After amplification, a sandwich hybridization assay was subsequently conducted within a PDMS channel [955]. 

In another report, real-time NASBA was used to amplify RNA in a Si-Pyrex chip containing 50-nL reaction chambers. This is an isothermal (41°C) technique. A fluorescent molecular beacon probe was used to detect the RNA derived from the human papillomavirus (HPV16) [337]. 

Purification of mRNA and subsequent single-strand cDNA synthesis by reverse transcription (RT) were achieved all on the same chip [956,957]. 

FIGURE 9.13 Schematic diagram of the microfabricated device for continuous-flow PCR and RT-PCR. There are two inlets for PCR (PCR1 and 2), two inlets for RT (RT1 and 2), and five outlets for product collection at 20, 25, 30, 35, and 40 cycles. The chip is placed over four heating blocks. Blocks a, b, and c are at appropriate temperatures for denaturation, extension, and annealing in PCR, respectively. Block d is for RT. The intersection between the RT and the PCR channels is denoted I [907]. Reprinted with permission from the American Chemical Society. 

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RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

Key Words Mieroarrays gene expression RNA amplification laser capture mierodisseetion eaneer stem eell theory pure cell populations diagnostics prognostics... 

Gallardo TD, Hammer RE, Garry DJ. RNA amplification and transcriptional profiling for analysis of stem cell populations. Genesis 2003 37 57-63. 

Artificially enhanced error rates needed for the creation of sequence diversity in a population can be achieved readily with PCR. Reverse transcription and transcription are also susceptible to increase of mutation rates. These two and other new techniques for RNA amplification provide universal and efficient tools for the study of molecular evolution under laboratory conditions and make the usage of viral replicases with their undesirable sequence specificities obsolete. 

Huang et al. [176] described an integrated microfluidic chip (of PDMS and soda-lime glass) capable of performing DNA/RNA amplification, electroki-netic sample injection and separation, and online optical detection in an automatic mode. The authors tested its functionality for bacterial DNA of Streptococcus pneumoniae and RNA of dengue-2 vims. The NCE developed represented a crucial contribution to the fields of molecular biology, genetic analysis, infectious disease detection, and other biomedical applications. 

Obeid, P.J., Christopoulos, T.K., Crabtree, H.J., Backhouse, C.J., Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection. Anal. Chem. 2003, 75, 288-295. 

Polymerase chain reaction The method for producing, in vitro and fairly rapidly, millions of copies of a specific segment of DNA or RNA (amplification). The sequence of the ends of the portion to be copied must be known so that the primer can be annealed to the denatured oligo to be copied. An excellent source for PCR applications is www.eppendorfsi.com/application.html. 

Instead of specific amplification of one target to improve sensitivity, methods that amplify all genomic DNA or mRNAs are useful when the target is in short supply. For example, multiple-displacement amplification uses exonuclease-resistant random hexamers and a highly pro-cessive polymerase to amplify DNA nonspecificaily. Initial DNA denaturation is not necessary and the reaction proceeds isothermally. Similarly, messenger RNA can be generi-caUy amplified with a poly(T) primer modified with an RNA polymerase promoter. After reverse transcription, second-strand DNA synthesis, and transcription, antisense RNA is produced. Both whole genome and antisense RNA amplification are also useful as nucleic acid purification methods before amplification or detection. 

Phillips J, Eberwine JH. Antisense rna amplification a linear amplification method for analyzing the mrna population from single living cells. Methods 1996 10 283-8. 

Steketee RW, Abrams EJ, Thea DM, Brown TM, Lambert G, Orloff S, et al. Early detection of perinatal human immunodeficiency virus (HIV) type 1 infection using HIV RNA amplification and detection. J Infect Dis 1997 175 707-11. 

When comparing hybridization results between two cell types, the same type and quantity of RNA should be used to generate both probes. If sufficient material is available, probes can be directly generated by incorporating fluorescent nucleotides during a reverse-transcription reaction. Otherwise an additional RNA amplification step will be needed. This is not desirable because it could cause problems with transcript representation. 

If only small quantities of RNA are available, then RNA amplification using T7 RNA polymerase to produce transcripts from double-stranded cDNA may be necessary (3). 

To allow efficient extraction of intact mRNA, it is recommended that alcohol fixatives be used in lieu of cross-linking agents such as paraformaldehyde. In our experience protocols that have been developed for studying paraformaldehyde-fixed paraffin-embedded archival specimens in tandem with RNA amplifications produce low yields of RNA from microdissected brain regions and picked cells. Fixation of tissue with methanol or ethanol preserves immu-noreactivity and mRNA in many cases. Accordingly, we immerse... 

Ginsberg SD, Che S (2004) Combined his-tochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus. Lab Invest 84 952-962... 

Although RT-PCR based methods are promising for RNA amplification, they require a DNAse step to remove DNA so that it is not coamplified along with RNA (68). Unfortunately, DNAse treatment has been reported to not be completely effective in this regard, thus additional complex steps are required (69). 

An alternative approach to pooling, in the analysis of small samples, is RNA amplification. In particular, this approach has been successfully used to derive enough RNA from sources such as laser capture microdissection... 

Fig. 2. (A) Agarose gel electrophoresis of total RNA (500ng/lane) extracted from four different regions (1-4) of the neural tube of E9.5 CDl mouse embryos using the column protocol detailed in section 3.3. The arrows point to the characteristic 28s and 18s rRNA bands. For intact total RNA, the 28s band should be approximately double the intensity of the 18s band see Notes 1-11). (B) Agarose gel electrophoresis of the biotin labeled extracts generated by the Ovation Biotin RNA Amplification and Labelling System (Enzo) from...

Labeled Extract Synthesis Ovation Biotin RNA Amplification and Labeling System (for lOng Total RNA)... 

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