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RNA Extraction & Purification

Isolating high-quality RNA is crucial to many downstream application, such as cloning, reverse transcription for cDNA synthesis, RT-PCR, RT-qPCR and RNA-seq. Isolation of total RNA from cells, blood, tissues and other samples can be accomplished using guanidium-phenol reagents followed by precipitation with lithium chloride and ethanol. Alternatively, commercial kits containing optimized buffer systems and silica columns can be employed.
 
Studies that require the isolation of eukaryotic mRNA often take advantage of the polyadenine tail typically present at the 3’ end. One approach involves the use affinity matrices; poly-T oligonucleotides that hybridize to the complementary poly-A tails (1). Enrichment of miRNA and siRNAs can also be achieved using high-affinity binding proteins and the use of chitin magnetic beads (2, 3).
 
Similar to DNA, enzymatic reactions in which RNA is used as a substrate may require cleanup steps for buffer exchange or removal of contaminants. Commercial RNA cleanup kits are also available for this application.
 

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