이화여대 생명과학실험 A+ 리포트(Realtime PCR을 이용한 유전자 발현 분석)

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1. RNA sequencing

RNA sequencing은 DNA로부터 전사되는 모든 RNA 전사체의 염기서열을 차세대 분석기술을 사용하여 분석하는 기술이다. RNA sequencing을 위해서는 cDNA가 필요한데 cDNA를 준비하는 일반적 과정은 보통 RNA 추출>RNA 선택>cDNA 합성>라이브러리 제작으로 이루어진다. RNA 염기서열 분석 기술을 통해 alternative RNA splicing, 유전자의 발현량, 염기서열의 변이, gene fusion, single nucleotide polymorphism, SNP 등의 정보를 얻을 수 있다.
2. Northern blot

분자생물학 연구에서 시료 내의 RNA를 탐지하는 방법을 이용함으로써 유전자 발현을 연구하기 위한 기술이다. 방법을 살펴보면 전기영동을 이용하여 크기별로 분리된 RNA 중 원하는 것을 찾아내는 것부터 시작한다. Northern blot은 다양한 상황에서 유전자의 발현을 조사하는 데 사용될 수 있는데, 수정란의 발생단계, 여러 세포나 조직과 기관, 병원균의 감염, 주변 환경에 의한 변화 등이 예가 된다.
3. cDNA 합성

역전사효소를 이용한 cDNA합성에서는 RNA와 Anealing 위치에 따라 세 종류의 프라이머를 사용할 수 있는데 Oligo dT primer, Random Hexamer primer, Gene-specific primer가 있다. Oligo dT primer는 mRNA의 POLY A tail에 anealing 되어, 3'말단에서부터 cDNA를 합성한다. Random Hexamer primer는 mRNA의 모든 영역에서 합성을 시작할 수 있다. Gene-specific primer는 특정한 target cDNA만을 합성할 수 있다.
4. Housekeeping gene

Housekeeping gene이란 세포의 생명활동 유지를 위해 어떤 조건에서도 높은 수준으로 발현되어 유지되는 유전자군을 의미한다. 즉 다른 유전자에 비해 환경 등 외부자극에 의해 큰 영향을 받지 않기 때문에 실험에서 비교,대조를 위해 활용되는 유전자이다.
5. Real-time PCR

Real-time PCR은 증폭 과정에서 우리가 관찰할 수 있는 것이 가능하고 DNA 초기양과 증폭된 양을 동시에 비교하는 것이 가능하다. 또한 real time PCR은 conventional PCR에 비해 극소량의 DNA도 증폭시킬 수 있기 때문에 검출감도가 높아 바이러스 DNA의 양을 검출하기 더 유리하다.

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1. RNA sequencing

RNA sequencing is a powerful technique that allows for the comprehensive analysis of the transcriptome, providing insights into gene expression patterns, alternative splicing, and the identification of novel transcripts. This technology has revolutionized our understanding of biological processes, enabling researchers to explore the complex dynamics of gene regulation and expression in various cell types and tissues. By generating high-resolution data on the RNA landscape, RNA sequencing has become an indispensable tool in fields such as developmental biology, cancer research, and personalized medicine. The ability to quantify transcript levels with high sensitivity and accuracy has opened new avenues for understanding disease mechanisms, identifying biomarkers, and developing targeted therapies. As the technology continues to evolve, with improvements in sequencing depth, read length, and computational analysis, RNA sequencing will undoubtedly remain a cornerstone of modern molecular biology and genomics research.
2. Northern blot

The Northern blot is a well-established technique in molecular biology that allows for the detection and quantification of specific RNA molecules within a complex sample. While it may not be as high-throughput as newer technologies like RNA sequencing, the Northern blot remains a valuable tool in the researcher's arsenal. Its ability to provide information on the size, abundance, and potential isoforms of target RNAs makes it a crucial complement to other RNA analysis methods. The Northern blot's strength lies in its ability to visualize and quantify the expression of specific genes, which is particularly useful in studies of gene regulation, developmental processes, and disease-associated changes in RNA expression. As newer techniques continue to emerge, the Northern blot will likely maintain its relevance, especially in situations where targeted, high-resolution analysis of individual transcripts is required. Its simplicity, robustness, and the wealth of existing knowledge and protocols make it an enduring technique in the field of molecular biology.
3. Housekeeping gene

Housekeeping genes are a crucial concept in molecular biology, as they play a fundamental role in the normalization and interpretation of gene expression data. These genes, which are typically involved in basic cellular processes and are expressed constitutively across various cell types and conditions, serve as reliable internal controls for techniques like qRT-PCR, Northern blotting, and RNA sequencing. The identification and use of appropriate housekeeping genes are essential for accurate quantification of target gene expression, as they help to account for variations in sample preparation, RNA extraction, and other experimental factors. The careful selection and validation of housekeeping genes are particularly important in studies where subtle changes in gene expression or the comparison of different biological conditions are the focus. As our understanding of the transcriptome and the complexities of gene regulation continue to evolve, the role of housekeeping genes in data normalization and interpretation will remain a critical aspect of modern molecular biology research.
4. Real-time PCR

Real-time PCR, also known as quantitative PCR (qPCR), is a powerful technique that has revolutionized the field of gene expression analysis. This method allows for the sensitive, specific, and quantitative detection of target DNA or RNA sequences in real-time, providing valuable insights into the expression levels of genes of interest. The ability to monitor the amplification of target molecules during the PCR reaction, coupled with the use of fluorescent probes or dyes, enables researchers to accurately quantify the initial amount of template present in a sample. Real-time PCR has become an indispensable tool in a wide range of applications, including gene expression profiling, pathogen detection, copy number variation analysis, and the validation of results obtained from high-throughput techniques like RNA sequencing. The continuous advancements in instrumentation, reagents, and data analysis software have further enhanced the sensitivity, specificity, and reproducibility of real-time PCR, making it a versatile and widely adopted technique in both research and clinical settings.