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Trace a picture. Draw curves and lines over the picture. You can then … is your solution. The program supports quadratic Bezier curves and cubic Bezier curves so that you can lay the curves smooth around the contours. You can edit the knots and the control points of the curves until the curve matches your ideas. You can.

Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

The study of gene expression in a cell or tissue at a particular moment gives an insight into the capacity of the cell for protein synthesis. Gene expression assays, for example, gene profiling, are an important tool and are widely used in nanotoxicity studies. There are several methods available to determine gene expression, such as northern blot analysis, ribonuclease protection assay (RPA), serial analysis of gene expression (SAGE), reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qRT-PCR), PCR arrays, and microarrays. Among these techniques, Northern blot analysis remains a standard method for detection and quantitation of mRNA levels despite the advent of more robust techniques. Northern blotting involves the use of electrophoresis to separate RNA samples by size, then detect the mRNA with a hybridization probe complementary to part of the target sequence. RPA is an extremely sensitive technique for the quantitation of specific RNAs in solution. It can be performed on total cellular RNA or poly(A)-selected mRNA as a target. SAGE method, as well as PCR arrays and microarrays, is used to study partial or global gene expression in cells or tissues in various experimental conditions. In this chapter, we will describe the methods to determine gene expression by using RT-PCR and real-time PCR. RT-PCR as a relatively simple, inexpensive, extremely sensitive and specific tool to determine the expression level of target genes. Real-time PCR is a quantitative method for determining copy number of PCR templates, such as DNA or cDNA, and consists of two types: probe-based and intercalator-based. Probe-based real-time PCR, also known as TaqMan PCR, requires a pair of PCR primers and an additional fluorogenic oligonucleotide probe with both a reporter fluorescent dye and a quencher dye attached. The intercalator-based (SYBR Green) method requires a double-stranded DNA dye in the PCR which binds to newly synthesized double-stranded DNA and generates fluorescence. Both methods require a special thermocycler equipped with a sensitive camera that monitors the fluorescence in each sample at frequent intervals during the PCR. The principle techniques underlying both RT-PCR and real-time PCR are total RNA isolation, reverse transcription (RT), and PCR. Reverse transcription involves the synthesis of DNA from RNA by using an RNA-dependent DNA polymerase. PCR can amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Here we will introduce detailed procedures for RT-PCR and real-time PCR.

Obtaining high quality and intact RNA is the first and often the most critical step in performing RT-PCR and real-time PCR. RNA is easily degraded since RNase is very hard to inactivate. Several precautions need to be taken to prevent RNA from degradation. People should always wear a clean lab coat, disposable gloves, and change gloves frequently. The bench should be clean. Any aqueous solutions, tubes, and pipettes used for the procedure should be sterile and RNAse-free. To avoid contaminating your sample with RNases, do not talk while processing RNA extraction.

Reverse transcription involves the synthesis of DNA from RNA by using an RNA-dependent DNA polymerase, the reverse of normal transcription, which is from RNA to DNA. Although there are many kits commercially available for RT, the reverse transcriptase used in those kits usually is M-MLV reverse transcriptase from the Moloney murine leukemia virus or AMV reverse transcriptase from the avian myeloblastosis virus. M-MLV reverse transcriptase is the preferred reverse transcriptase in cDNA synthesis for long messenger RNA (mRNA) templates (>5 kb) because the RNase H activity of M-MLV reverse transcriptase is weaker than the commonly used AMV reverse transcriptase (2). Since M-MLV reverse transcriptase is less processive than AMV reverse transcriptase, therefore, more units of the M-MLV enzyme are required to generate the same amount of cDNA as in the AMV reaction (2). The following are the basic procedures for RT using M-MLV reverse transcriptase according to the manufacturer’s instruction.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase. There are many DNA polymerases commercially available. Although their efficiency may be different, they are suitable for regular RT-PCR to determine the expression level of mRNA. Some experiments which will use PCR products for cloning purposes, especially those for cloning of promoter region with high G-C content, need to use high fidelity DNA polymerase. The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The following is an example of a PCR performed in our laboratory.

Traditional PCR uses agarose gel for detection of PCR amplification at the final phase or endpoint of the PCR (plateau). However, real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction. Theoretically, there is a quantitative relationship between the amount of starting target sample and the amount of PCR product at any given cycle number. Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR, which makes quantitation of DNA and RNA easier and more precise than traditional methods. There are two methods, which are often used in the laboratory. One is the 5′ nuclease assay in which an oligonucleotide called a TaqMan® Probe is added to the PCR reagent master mix. This probe is designed to anneal to a specific sequence of template between the forward and reverse primers and is also designed with a high-energy dye termed a Reporter at the 5′ end, and a low-energy molecule termed a Quencher at the 3′ end. When this probe is intact and excited by a light source, the Reporter dye’s emission is suppressed by the Quencher dye as a result of the close proximity of the dyes. When the probe is cleaved by the 5′ nuclease activity of the enzyme, the distance between the Reporter and the Quencher increases causing the transfer of energy to stop. The fluorescent emissions of the reporter increase and the quencher decrease. An increase in Reporter fluorescent signal is directly proportional to the number of amplicons generated. Another real-time PCR method is by using SYBR Green Dye, which can bind the minor groove of any double-stranded DNA molecule. When SYBR Green dye binds to double-stranded DNA, the intensity of the fluorescent emissions increases. As more double-stranded amplicons are produced, SYBR Green dye signal will increase. The following is an example of real-time PCR by using iQ™ SYBR Green Supermix and performed on an iQ5 multicolor real-time PCR detection system.

Currently, there are a number of RNA isolation kits commercially available. Although it is convenient, time-saving, and avoids contact with phenol/chloroform using commercially available kits, those kits using silica-membrane spin columns may not be ideal for studies of insoluble nanoparticle since the nanoparticles may clog the membrane pore of the spin column. Therefore, it is important to choose the right reagents or kits for total RNA isolation according to different experiments and specific characteristics of different nanoparticles. In our laboratory, we use TRI Reagent to isolate total RNA for nanoparticle studies. TRI Reagent is a mixture of guanidine thiocyanate and phenol in a monophase solution, which can effectively dissolve DNA, RNA, and protein after homogenization or lysis of tissue samples. It performs well with large or small amounts of tissue or cells. Here, we describe the procedures for isolating total RNA using TRI Reagent according to the manufacturer’s instructions.

 

Curves are a very versatile and precise color correction … accuracy to take full advantage of it. Fresh Curves makes up for these drawbacks so you can … of this basic tool. More channels Fresh Curves has brightness, saturation and hue channels for more … on top of the usual RGBA. Relative Curves Now it’s possible to adjust on attributes based and spirographs. Written in Java and rendered using Bezier curves. The editor supports multiple configurable objects superimposed on … In all cases, the shapes are rendered using Bezier curves, which are used to approximate the true shape.

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