Isolating high-quality RNA molecules is crucial to many downstream experiments, such as cloning, reverse transcription for cDNA synthesis, RT-PCR, RT-qPCR and RNA-seq. There are various approaches to RNA purification including phenol-chloroform extraction, spin column purification, and the use of magnetic beads.
Total RNA purification involves the extraction and purification of total RNA from your sample, for use in gene expression analyses such as RT-qPCR or RNA-seq. Depending on the starting sample, different protocols should be employed to most efficiently lyse cells or tissues, and purify the RNA from contamination genomic DNA and other cellular components. Theoretically, total RNA purification involves the isolation of all of the cellular RNA, including various sizes ranging from intact ribosomal RNA down to small RNA (including miRNA, tRNA and 5sRNA).
For recovery of a specific RNA subpopulation from total RNA derived from cells, tissues or in vitro transcriptions, bead-based methods may be utilized to isolate mRNAs through binding of the poly(A) tail or to isolate RNAs based on size by varying the ratio of nucleic acid to beads in the presence of a crowding reagent. Bead-based methods are a rapid way to isolate RNA free from enzymes, salts and nucleotides from a variety of sources, and when RNA is present at lower concentrations.
In vitro transcribed RNA also often needs to be cleaned up and separated from components such as DNA templates, unincorporated nucleotides or RNA modifying enzymes. Chemical-, column- and gel-based approaches can be used, and are sometimes combined to result in purified RNA free from nucleotides, salts and proteins. Learn more about cleaning up in vitro transcribed RNA.
Regardless of the methods employed, purified RNA is ready for use in various downstream applications including expression analysis, transient transfections or microinjections to assess genome editing or RNA localization, as well as in vitro assays including RNA structure studies, nucleic acid hybridizations and in vitro translation.
- Is the Monarch Total RNA Miniprep Kit compatible with Luna RT-qPCR Reagents?
- Is the Monarch Total RNA Miniprep Kit compatible with NEBNext reagents for RNA Library Prep?
- What is the lowest volume in which I can elute my RNA with the Monarch Total RNA Miniprep Kit (NEB #T2010)?
- Can I purchase Monarch buffers and columns separately?
- Can I skip the gDNA removal column step of the Monarch Total RNA Miniprep Kit protocol?
- Can I use NEB DNase I M0303 with the Monarch Total RNA Miniprep Kit?
- Can I use the Monarch Total RNA Miniprep Kit for RNA cleanup after enzymatic manipulations?
- Can the Monarch Total RNA Miniprep Kit be used to purify RNA from plants?
- Can the Monarch Total RNA Miniprep Kit be used to purify RNA from yeast?
- How can I preserve my sample if I am not ready to extract RNA right after harvest?
- I am seeing genomic DNA in my preps when using the Monarch Total RNA Miniprep Kit What could be the problem?
- Can I lyse my adherent cells directly in their wells when using the Monarch Total RNA Miniprep Kit?
- Batch Method Protocol (S1560) for Isolation of mRNA Using the PolyA Spin™ mRNA Isolation Kit
- cDNA synthesis on oligo (dT)25 magnetic beads (S1419)
- Isolation of mRNA from Mammalian Cells Using the Magnetic mRNA Isolation Kit (NEB #S1550)
- Isolation of mRNA from Tissue Using the Magnetic mRNA Isolation Kit (NEB #S1550)
- Isolation of mRNA Using the PolyA Spin™ mRNA Isolation Kit
- Protocol for isolation of siRNA from p19 siRNA Binding Protein (M0310) using chitin magnetic beads
- Quantitation of Isolated Poly(A) RNA from the Magnetic mRNA Isolation Kit (NEB #S1550)
- Isolate mRNA using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490)
- Ligation protocol using SplintR® Ligase (M0375)
- Protocol for Avoiding Rnase Contamination using Rnase Inhibitor, Human Placenta (M0307)
- RNA Extraction from Blood
- RNA Extraction from Cells
- Total RNA Purification from Samples Stored in Monarch DNA/RNA Protection Reagent using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- RNA Extraction from Tissues
- Total RNA Purification from Tough-to-Lyse Samples (bacteria, yeast, plant, etc.) using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Fractionation of Small and Large RNA Using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- RNA Reaction Cleanup using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Total RNA Purification from Samples in RNAlater® using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Total RNA Purification from TRIzol®-extracted Samples using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- In-tube DNase I Treatment after Purification with the Monarch Total RNA Miniprep Kit (NEB #T2010)
- FFPE Tissue Deparaffinization and Subsequent RNA Purification Using the Monarch Total RNA Miniprep Kit NEB T2010
- Quick Protocol for Total RNA Extraction (NEB #T2010)
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