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Monocle [121] Differential expression and time-series analysis for single-cell RNA-Seq and qPCR experiments. SCANPY [122] [123] Scalable Python-based implementation for preprocessing, visualization, clustering, trajectory inference and differential expression testing. SCell [124] integrated analysis of single-cell RNA-seq data.
If validation of transcript isoforms is required, an inspection of RNA-Seq read alignments should indicate where qPCR primers might be placed for maximum discrimination. The measurement of multiple control genes along with the genes of interest produces a stable reference within a biological context.
RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome. [2] [3]
In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction (PCR) is a common method for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to complementary DNA (cDNA) with reverse transcriptase.
The close association between RT-PCR and qPCR has led to metonymic use of the term qPCR to mean RT-PCR. Such use may be confusing, [2] as RT-PCR can be used without qPCR, for example to enable molecular cloning, sequencing or simple detection of RNA.
The MIQE guidelines were created due to the low quality of qPCR data submitted to academic journals at the time, which was only becoming more common as Next Generation Sequencing machinery allowed for such experiments to be run for a cheaper cost. Because the technique is utilized across all of science in multiple fields, the instruments ...
An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq. [1] A spike-in is designed to bind to a DNA molecule with a matching sequence, known as a control probe. [2] [3] [4] This process of specific binding is called ...
The development of high-throughput RNA sequencing (RNA-seq) and microarrays has made gene expression analysis a routine. RNA analysis was previously limited to tracing individual transcripts by Northern blots or quantitative PCR. Higher throughput and speed allow researchers to frequently characterize the expression profiles of populations of ...
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