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Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

5.9K
Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Sanger Sequencing01:57

Sanger Sequencing

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DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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Next-generation Sequencing03:00

Next-generation Sequencing

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
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RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Ribosome Profiling02:24

Ribosome Profiling

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
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Video Experimental Relacionado

Updated: May 5, 2026

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
11:52

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Published on: August 4, 2016

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Secuenciación directa de ARN por secuenciación directa de ARN.

Fatih Ozsolak1, Adam R Platt, Dan R Jones

  • 1Helicos BioSciences Corporation, One Kendall Square, Cambridge, Massachusetts 02139, USA. fatihozsolak@gmail.com

Nature
|September 25, 2009
PubMed
Resumen
Este resumen es generado por máquina.

La secuenciación directa de ARN evita la conversión del ADN complementario (ADNc), reduciendo los sesgos y mejorando el análisis de transcripción. Este método innovador permite la secuenciación directa de ARN de alto rendimiento y bajo costo para una comprensión completa de los transcriptomas.

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Área de la Ciencia:

  • Genómica y transcriptómica.
  • Biología Molecular Biología Molecular
  • La bioinformática es la bioinformática.

Sus antecedentes:

  • La comprensión del genoma y el transcriptoma es crucial para la biología humana y la investigación de enfermedades.
  • El análisis actual del transcriptoma se basa en la conversión del ADN complementario (ADNc), introduciendo sesgos y limitando el análisis del ARN degradado o de baja cantidad.
  • Los métodos existentes impiden una comprensión completa de la transcripción de todo el genoma y el estado dinámico del ARN.

Objetivo del estudio:

  • Desarrollar y aplicar un método directo de secuenciación de ARN de una sola molécula sin previa conversión de ARN a ADNc.
  • Para superar las limitaciones de los métodos actuales de análisis del transcriptoma, incluidos los sesgos y la idoneidad para el ARN degradado.
  • Para permitir la secuenciación directa de ARN de alto rendimiento, bajo costo y sin sesgo para una comprensión completa del transcriptoma.

Principales métodos:

  • Se realizó la secuenciación directa de ARN de una sola molécula sin convertir el ARN en ADNc.
  • Se secuenciaron cantidades femtomólicas de ARN de Saccharomyces cerevisiae poliadenilado [poly (((A) (((+) ].
  • El ARN se capturó utilizando una superficie recubierta con oligonucleótidos poli (dT), iniciando la secuenciación por síntesis a través de colas naturales de poli (A).

Principales resultados:

  • Secuenciado con éxito ARN directamente sin conversión de ADNc, lo que demuestra un nuevo enfoque.
  • Se observó la heterogeneidad del extremo 3' del transcrito, lo que proporciona información sobre el procesamiento del ARN.
  • Identificó ARN nucleolares pequeños poliadenilados, ampliando el conocimiento de las poblaciones de ARN no codificantes.

Conclusiones:

  • La secuenciación directa de ARN ofrece un camino hacia un análisis de transcriptoma de alto rendimiento y rentable.
  • Este método minimiza los sesgos asociados con la síntesis de ADNc, permitiendo una caracterización y cuantificación de transcripciones más precisas.
  • Alcanza el objetivo de una comprensión integral y libre de prejuicios de los transcriptomas, crucial para la investigación biológica y de enfermedades.