[생물공정실험] 2주차 RNA extraction and quantification 예비보고서

본 내용은 " [생물공정실험] 2주차 RNA extraction and quantification 예비보고서 " 의 원문 자료에서 일부 인용된 것입니다. 2023.01.16

문서 내 토픽

• 1. Gene Expression

  유전자 발현은 유전자에 암호화된 정보가 단백질을 암호화하는 RNA를 만들거나 다른 기능을 수행하는 비암호화 RNA를 만드는데 사용되는 과정이다. RNA와 단백질이 만들어지는 시기와 장소를 제어하는 스위치 역할을 하고 산물이 얼마나 많이 만들어지는지 결정하는 역할을 한다. DNA에서 RNA로 전사, 단백질로의 번역을 통해 세포에 존재하는 단백질의 양을 결정하게 된다. 세포에 있는 mRNA 분자의 양과 유형은 세포의 기능을 나타낸다. RNA 전사는 단일 mRNA 분자에서 많은 단백질을 만들 수 있기 때문에 효율적인 제어 지점이 된다.

• 2. RNA Purification

  RNA를 분리하는 것은 cloning, cDNA 합성을 위한 역전사, RT-PCR, RT-qPCR과 같은 많은 다운스트림 실험에 매우 중요하다. Phenol-Chloroform 추출, spin column 정제와 같이 RNA 정제에 다양한 접근 방식이 있다. 리보솜 RNA나 tRNA는 일정한 크기와 염기서열을 가지고 있기 때문에 전기영동, 초원심분리 및 크로마토그래피에 의해 순수하게 분리할 수 있는 반면 mRNA는 크기와 종류가 다양해 순수하게 분리해내기 어렵다. 하지만 진핵세포 mRNA는 3말단에 polyA를 가져 rRNA나 tRNA등으로부터 분리해낼 수 있다.

• 3. Trizol RNA Purification

  Trizol은 RNA, DNA, protein을 분리할 때 사용하는 시약으로 단상용액에 guanidium isothiocyanate와 phenol을 섞은 혼합물이고 Rnase를 억제한다. Trizol로 샘플 homogenization을 실시한 후 chloroform을 추가하면 RNA가 함유된 맑은 상층액과 DNA 및 단백질이 들어있는 중간층, 아래 붉은색 유기층으로 분리된다. RNA는 상층액에 isopropanol을 넣어 침전시킬 수 있다.

• 4. RNA Purity Analysis

  DNA, RNA의 농도 및 순도를 측정하기 위해 nano-drop spectrophotometer를 사용해 광학 밀도를 측정한다. A260/280 ratio가 1.8~2.1이면 좋은 순도를 나타내며, A260/230 ratio가 1.9~2.2이면 좋은 순도를 나타낸다. 이를 통해 RNA가 Rnase free water에 얼마나 용해되어 있는지, total RNA의 양을 확인할 수 있다.

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• 1. Gene Expression

  Gene expression is a fundamental process in biology that involves the conversion of genetic information encoded in DNA into functional proteins or other molecules. It is a complex and highly regulated process that plays a crucial role in the development, growth, and maintenance of all living organisms. Understanding gene expression is essential for various fields, including medicine, biotechnology, and genetics, as it provides insights into how cells and organisms respond to different stimuli, environmental conditions, and genetic factors. By studying gene expression, researchers can gain valuable knowledge about the underlying mechanisms of biological processes, identify potential therapeutic targets for diseases, and develop new strategies for genetic engineering and personalized medicine.

• 2. RNA Purification

  RNA purification is a crucial step in many molecular biology and biotechnology applications, as it allows for the isolation and extraction of high-quality RNA molecules from various biological samples. Accurate and efficient RNA purification is essential for downstream analyses, such as gene expression studies, RNA sequencing, and reverse transcription-PCR (RT-PCR). The choice of RNA purification method depends on the sample type, the intended application, and the required purity and yield of the RNA. Common RNA purification techniques include column-based methods, phenol-chloroform extraction, and magnetic bead-based approaches. Each method has its own advantages and limitations, and the selection of the appropriate technique is crucial to ensure the integrity and quality of the extracted RNA. Proper RNA purification is a fundamental step in many research and diagnostic applications, and continued advancements in this field will contribute to the advancement of various scientific and medical disciplines.

• 3. Trizol RNA Purification

  Trizol (also known as TRIzol) is a widely used reagent for the purification of RNA from a variety of biological samples, including cells, tissues, and organisms. The Trizol RNA purification method is based on the single-step RNA isolation technique developed by Chomczynski and Sacchi in the 1980s. This method combines the guanidinium thiocyanate-phenol-chloroform extraction with the selective precipitation of RNA to isolate high-quality, intact RNA. The Trizol reagent effectively lyses cells and denatures proteins, allowing for the efficient separation of RNA from DNA and proteins. The Trizol RNA purification protocol is relatively simple, cost-effective, and can be used to isolate RNA from a wide range of sample types. However, it is important to follow the protocol carefully and to use high-quality reagents to ensure the purity and integrity of the extracted RNA. Proper Trizol RNA purification is a crucial step in many molecular biology and genomics applications, and its continued use in research and diagnostic settings highlights its importance in the field of RNA analysis.

• 4. RNA Purity Analysis

  Analyzing the purity of extracted RNA is a critical step in many molecular biology and biotechnology applications, as the quality and integrity of the RNA can significantly impact the accuracy and reliability of downstream analyses. The purity of RNA is typically assessed by measuring the absorbance ratios at different wavelengths, such as the A260/A280 and A260/A230 ratios. The A260/A280 ratio provides an estimate of the protein contamination in the RNA sample, while the A260/A230 ratio indicates the presence of other contaminants, such as phenol, carbohydrates, or guanidinium salts. Ideally, the A260/A280 ratio should be between 1.8 and 2.0, and the A260/A230 ratio should be greater than 2.0, indicating a high-purity RNA sample. In addition to spectrophotometric analysis, other techniques, such as agarose gel electrophoresis or microfluidic-based platforms (e.g., Bioanalyzer), can be used to assess the integrity and quality of the extracted RNA. Proper RNA purity analysis is crucial for ensuring the reliability and accuracy of downstream applications, such as gene expression studies, RNA sequencing, and RT-PCR. Continued advancements in RNA purity assessment methods will contribute to the advancement of various fields in molecular biology and biotechnology.