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.
FAQs for RNA Purification and Isolation
- Is the Monarch® Total RNA Miniprep Kit compatible with NEBNext® reagents for RNA Library Prep?
- Is the Monarch® Total RNA Miniprep Kit compatible with Luna® RT-qPCR Reagents?
- Can I skip the gDNA removal column step of the Monarch® Total RNA Miniprep Kit protocol?
- Can I use the Monarch® Total RNA Miniprep Kit for RNA cleanup after enzymatic manipulations?
- Can I use NEB DNase I (#M0303) with the Monarch Total RNA Miniprep Kit?
- I am seeing genomic DNA in my preps when using the Monarch Total RNA Miniprep Kit. What could be the problem?
- How can I preserve my sample if I am not ready to extract RNA right after harvest?
- Can the Monarch® Total RNA Miniprep Kit be used to purify RNA from plants?
- What is the lowest volume in which I can elute my RNA with the Monarch Total RNA Miniprep Kit (NEB #T2010)?
- Can I lyse my adherent cells directly in their wells when using the Monarch Total RNA Miniprep Kit?
- Can the Monarch® Total RNA Miniprep Kit be used to purify RNA from yeast?
- Can I purchase Monarch® buffers and columns separately?
Protocols for RNA Purification and Isolation
- Batch Method Protocol (S1560) for Isolation of mRNA Using the PolyA Spin™ mRNA Isolation Kit
- cDNA synthesis in oligo (dT)25 magnetic beads (S1419)
- FFPE Tissue Deparaffinization and Subsequent RNA Purification Using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Fractionation of Small and Large RNA 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)
- Isolate mRNA using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490)
- 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
- Ligation protocol using SplintR® Ligase (M0375)
- Protocol for Avoiding Rnase Contamination using Rnase Inhibitor, Human Placenta (M0307)
- 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)
- Quick Protocol for Monarch® Total RNA Miniprep Kit (NEB #T2010)
- RNA Reaction Cleanup using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Total RNA Purification from Cultured Mammalian Cells using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Total RNA Purification from Mammalian Whole Blood Samples 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 Samples Stored in Monarch DNA/RNA Protection Reagent using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Total RNA Purification from Tissues and Leukocytes using the Monarch Total RNA Miniprep Kit (NEB #T2010)
- Total RNA Purification from Tough-to-Lyse Samples (bacteria, yeast, plant, etc.) 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)
Latest NEB Expressions
This edition of NEB expressions focuses on tools that can streamline your RNA-related workflows. Featured products include NEBNext Ultra II kits for RNA, the Monarch Total RNA Miniprep Kit, Luna reagents for RT-qPCR, the new LunaScript RT SuperMix Kit and HiScribe kits for high yield, high quality RNA synthesis. Learn more about functional considerations for RNA synthesis in our feature, and learn about a new protocol for Cappable Seq. Lastly, read about ways you can promote sustainable science in the lab.
RNA Technical Guide
Find in depth information on tools designed to streamline your RNA workflows, including RNA extraction & purification, qPCR and RT-qPCR, RNA-seq, RNA synthesis and gRNA synthesis.
- Troubleshooting Guide for RNA Cleanup
Other Tools & Resources
Reagents for RNA Sample Preparation
RNA Polymerase Selection Chart
RNA Ligase Selection Chart
cDNA Synthesis Selection Chart
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For more information about commercial rights, please contact NEB's Global Business Development team at firstname.lastname@example.org.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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