RT-PCR reaction

One should avoid the use of highly cross-linking fixatives, such as mercuric chloride, glutaraldehyde, modified formalin, and picric-acid-based fixatives. These extensively cross-linking fixatives render the tissue virtually impermeable to the RT-PCR reaction components and to the probe. 

Add 30 0 pL of probe containing hybridization solution to each tissue section treated with the in situ RT-PCR reaction protocol. Cover with siliconized glass coverslips, seal with rubber cement. 

In 1995, Perkin-Ehner introduced a new enzyme rTth-DNA polymerase with a dual activity. It can perform both RT and PCR in the presence of manganese acetate buffer, sense and antisense primers, and nucleotides. This protocol is easier to perform and reduces total in situ RT-PCR reaction time (46). 

DEPC-treated water should be used for all RNA preparation solutions, gloves should be worn at all times, and RNase-free tips and tubes used. It is not necessary to purify mRNA, since total RNA prepared from freshly isolated or even frozen (liquid nitrogen or-80°C) lymphocytes is pure enough to carry out the RT-PCR reactions. A number of commercial kits are now available for RNA isolation (e.g., Stratagene), and use of one of these is recommended. Alternatively the single-step guanidimum thiocyanate/phenol extraction method of Chomczynski and Sacchi (23), on which most kit protocols are based, should be employed. If the lymphocyte numbers are low (<5 x 106), carrier RNA (100 pg of 16S ribosomal RNA) can be added at the start of this procedure to aid recovery. The preparation ends with a precipitation step using isopropanol, and the RNA may stored at -20°C in this form until required. 

For most purposes, contamination from genomic DNA can be eliminated in the RT-PCR reaction by designing primers that span an intron. The size difference between PCR products generated from genomic DNA and mRNA will preclude contamination. 

Single-strand cDNA that was reverse-transcribed from human hippocampal poly A+ RNA was used for RT-PCR reactions. 

To recover and amplify the genetic material after selection, a single-step RT-PCR is performed on ribosome-bound mRNA (Figure 1). Since the ribosome occupies the 3 end of the mRNA at the termination of translation, a downstream primer D2, designed to hybridize at least 60 nt upstream of the 3 end of the mRNA, is used in combination with the upstream primer T7Ab/back in the RT-PCR reaction (Figure 2 and Toble 2). Since the use of D2 produces a shortened DNA... 

Quantitative polymerase chain reaction, also called real-time RT-PCR or QPCR, is a method which employs insertion of a signal, such as fluorescence or enzyme activity, into PCR products generated by RT-PCR to determine the amount of messenger RNA (mRNA) in a tissue accurately. 

RT-PCR reverse transcriptase polymerase chain reaction RTV ritonavir... 

Cronin Breast cancer MasterPure TaqMan reactions RT-PCR analysis... 

Combining both heating and nonheating protocols employed in a sequential order were evaluated, but without any advantage (Fig. 3.4). RT-PCR was performed by standard methods, RNA extracted from fresh MDA cells and human tissue of breast cancer with known tested genes was used as positive control, and pure water was used to replace template (cDNA) as negative control for every experiment of PCR. To assure the accuracy of PCR tests, all reactions were performed in triplicate. 

Mies C. A simple, rapid method for isolating RNA from paraffin-embedded tissues for reverse transcription-polymerase chain reaction (RT-PCR). /. Histochem. Cytochem. 1994 42 811-813. 

Cyanine dyes also are used as labels for oligonucleotide probes. Unlike the hydrophilic cyanine dyes valuable for protein labeling, the use of dye-phosphoramidite compounds to synthesize DNA or RNA probes typically requires the use of more hydrophobic dye structures to make them compatible with the solvents and reactions of oligonucleotide synthesis. Thus, indol cyanines containing few or no sulfonates are used in these applications to label oligos for applications such as array detection, hybridization assays, and RT-PCR. 

Cytokine profiling has also been measured as a function of changes in cytokine mRNA expression using either reverse transcription polymerase chain reaction (RT-PCR) [87, 91-93] or ribonuclease protection assay (RPA) [94-97], Measurement of cytokine transcripts by RT-PCR revealed that prolonged exposure to TMA induced increased levels of IL-4 mRNA expression compared with treatment with DNCB [87,92-93]. However, expression of the type 1 cytokine IFN-y by DNCB-activated LNC was variable and failed to discriminate between contact and respiratory allergens [87,91,93). A similar profile was observed for freshly isolated tissue analyzed by RPA. This somewhat less... 

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

---