Measurement and Detection
it is possible to measure the transfer, it was found in a variety of ways. Measure the richness absolute nuclear RNA levels or total: to measure the relative abundance of chips chips RNAP and transcript RNase protection analysis newly formed: run in the analysis of nuclear detect active transcription site RT-PCR was but, DNA microarray may be different and the transfer rate: measuring the relative abundance of nuclear RNA levels or the whole world, but they may in situ hybridization rate of transcription different: detect the presence of MS2 marking transcript is: by including the MS2 RNA stem and lines, is a RNA which is synthesized de novo in the gene, they are incorporated. Stem line can be detected using the MS2 coat protein having the sequence specific interaction with high affinity GFP fusions and the trunk MS2. Recruitment of GFP to the site of transcription is visualized as a fluorescent spot. This new approach revealed to be generated by the pulse transfer or discontinuous burst. In the notable exception of location technology, many other ways to provide the average cell population, they can not detect the basic properties of the gene. Northern blot: RNA-SEQ, and how traditional appearance of most quantitative Next generation sequencing techniques transcriptome entire sequence can be used to measure the relative amount of RNA, to detect gene fusion such variations additional and new splice sites change or after transfer: RNA-SEQ apply
The (RT-PCR), which is one of many embodiments of the polymerase chain reaction (PCR) Reverse transcription-polymerase chain reaction. This technique is used in the field of molecular biology for detecting the expression levels of RNA widely. RT-PCR has been confused real-time polymerase chain reaction and scholars and students (QPCR) often. However, they are different techniques clearly. By creating a (cDNA) the additional DNA from RNA transcription, RT-PCR, are used for the qualitative detection of gene expression but, QPCR was quantified amplification of DNA using the fluorescent probe . Quantitative PCR, quantitative real-time PCR, , QPCR also serves as a real-time quantitative PCR. Conventional PCR and RT-PCR, by amplifying the application of the two techniques in this way, to generate multiple copies of DNA isolation of specific, but is fundamentally different. Conventional PCR is used merely for the exponential amplification of DNA sequences. RT-PCR was used to using reverse transcriptase, cloning a gene that is expressed by reverse transcription of RNA of interest in the complement of the DNA. The cDNA then was newly synthesized was amplified using conventional PCR.
In addition to the qualitative study of gene expression by including an equivalent QPCR, RT-PCR may be used to quantify the RNA absolutely and relatively. Often, combined technique described by (real-time quantitative RT-PCR sometimes) Quantitative RT-PCR, real time RT-PCR is an abbreviated RRT-PCR QRT-PCR, or RT-QPCR,. Most powerful compared to other methods for quantitative RNA by Northern blotting, detecting the level of the QRT-PCR RNA, can be considered quantitative analysis with high sensitivity. Infection and expression of one or more genes and this is used to analyze the expression patterns for the identification of disease often.
Quantification of mRNA using RT-PCR may be achieved either two-step reaction or one step. Difference between the two approaches is the number of tubes used in the course of the procedure. The approach of one stage, all of the cDNA synthesis reaction by PCR amplification occurs in the tube. On the other hand, require a reverse transcriptase reaction, the two-stage reaction was performed in a separate tube PCR amplification. The one-step method, I thought to minimize the deviation from the experiment of the enzyme reaction of all in the environment. However, it is susceptible to degradation in the approach of a single step, and the use of this approach, starting template RNA does not recommend that you repeated analysis of the same sample. Furthermore, the one-step approach, it is less accurate than a two-step approach was reported. The main advantage of the two-step approach, analysis with high reproducibility shows a correlation coefficient in the range of 0988-0974. Removal of primer dimers, so can be achieved by simple modification of the melting temperature, for example, be used DNA binding dye SYBR Green or the like, it is the preferred method of analysis. A disadvantage of two-stage approach is sensitive to contamination by more frequent handling samples.
Serum prostate-specific antigen (PSA) assay has a high false positive rate. Marker PSA is to provide diagnostic information is limited. As well as tumor, multi-marker test for detection of potentially fatal, provides a clinical need that is not met. Using the nanoString nCounter system, 20 – as a means for detecting prostate cancer, gene expression of the gene multiple test was developed based on the digital count gene RNA transcripts urine. In this test, centrifuged to pellet void urine, cells were amplified preselected hybridization probe sets the RNA was purified. Measured by DNA microarray, amplification test cell line RNA is not a gene rich level and the number of matches and bias considerable, but will be displayed. For data analysis, each number, to compare β2 microglobulin, and gene family. I have analyzed urine samples of five preoperative patients, and 2 non-cancer. Pathology information is restored. The low June 06 and Gleason scores and prostate cancer markers noncancerous low, are known, the signal for the majority of genes, were positive when. In contrast, one instance Gleason 4 Figure 5 is a strong signal for all associated with cancer markers including CD24. The non-cancer, there is shown a signal of cancer marker of 6 all, there is a possibility that undiagnosed, harbor cancer this person. Analyze the natural wastes, this multiplex assay can be used to stratify patients and early detection potential screening of cancer. The diagnostic information, I got from RNA signature associated with the cells of prostate cancer.