[생명공학] RNA extraction, cDNA synthesis, qRT PCR

문서 내 토픽

1. RNA extraction

RNA를 분리하는 과정에서 가장먼저 첨가해주는 물질이 Trizol이다. Trizol은 phenol과 guanidinium thiocyanate라는 두가지 중요한 성분으로 이루어져있고 이들이 cell을 lysis하고 RNA를 안정화시키는 역할을 한다. 추가적으로 chloroform을 첨가하여 RNA와 다른 물질들을 분리한다.
2. cDNA synthesis

cDNA 합성 과정에서는 reverse transcriptase 효소가 핵심적인 역할을 한다. 이 효소는 RNA를 DNA로 역전사하는 기능을 가지고 있다. 사용되는 primer에는 random primer, oligo(dT), target-specific primers 등이 있으며 각각의 장단점이 있다.
3. qRT-PCR

qRT-PCR은 probe로 표지된 DNA를 증폭시키면서 발생하는 형광의 양을 통해 초기 DNA의 양을 정량하는 방법이다. 이때 형광 signal이 특정 값을 넘게 될때의 cycle 수를 CT value라고 하고 이 CT value를 통해 정량분석을 할 수 있다. 하지만 PCR inhibition 문제로 인해 완벽한 정량은 어려울 수 있다.

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1. RNA extraction

RNA extraction is a critical step in many molecular biology and genomics experiments. It involves isolating and purifying RNA molecules from biological samples, such as cells, tissues, or organisms. This process is essential for downstream applications like gene expression analysis, RNA sequencing, and reverse transcription-PCR (RT-PCR). The quality and integrity of the extracted RNA are crucial for obtaining reliable and accurate results. There are various methods and commercial kits available for RNA extraction, each with its own advantages and limitations. The choice of method depends on factors like the sample type, RNA yield and purity requirements, and the downstream applications. Proper handling and storage of the extracted RNA are also important to prevent degradation. Overall, RNA extraction is a fundamental technique that enables researchers to study gene expression, understand cellular processes, and investigate various biological phenomena.
2. cDNA synthesis

cDNA synthesis, also known as reverse transcription, is a crucial step in many molecular biology and genomics experiments. It involves the conversion of RNA molecules into their complementary DNA (cDNA) counterparts, which can then be used for various downstream applications, such as gene expression analysis, cloning, and next-generation sequencing. The process of cDNA synthesis is typically carried out using the enzyme reverse transcriptase, which uses an RNA template to synthesize a single-stranded DNA molecule. The resulting cDNA can then be amplified using techniques like PCR or used as a template for further analysis. Accurate and efficient cDNA synthesis is essential for obtaining reliable and reproducible results, as it can impact the sensitivity, specificity, and quantitative accuracy of downstream experiments. Factors like RNA quality, primer design, and reaction conditions can all influence the success of cDNA synthesis. Proper optimization and quality control measures are necessary to ensure the integrity and fidelity of the cDNA generated.
3. qRT-PCR

Quantitative reverse transcription-PCR (qRT-PCR) is a powerful technique used for the sensitive and accurate quantification of gene expression levels. It combines the reverse transcription of RNA into cDNA with the amplification and detection of specific DNA sequences using real-time PCR. qRT-PCR allows researchers to measure the relative or absolute abundance of target mRNA transcripts in a sample, providing insights into gene regulation, cellular processes, and disease-related changes in gene expression. The technique relies on the use of fluorescent dyes or sequence-specific probes to monitor the amplification of the target cDNA in real-time, enabling the quantification of gene expression levels with high sensitivity and precision. Proper experimental design, including the selection of appropriate reference genes, primer and probe design, and data analysis methods, is crucial for obtaining reliable and meaningful results from qRT-PCR experiments. Additionally, the careful optimization of reaction conditions and the inclusion of appropriate controls are essential to ensure the accuracy and reproducibility of qRT-PCR data. Overall, qRT-PCR is a widely used and indispensable tool in molecular biology, genetics, and biomedical research.