RNA extraction

A full suite of products for all your RNA extraction needs

RNA extraction products comprise a full suite of kits and reagents that offer excellent reproducibility in many downstream molecular biology applications, including real-time RT-PCR, microarray analysis, next-generation sequencing (RNA-Seq), northern blotting, and cloning. Our RNA extraction products are optimized to provide maximum yield, purity, and integrity in an array of format options.

Browse by Application

RNA Extraction for Real-Time PCR

RNA Microarray
Analysis

In Vitro Transcription & Translation

Northern Blot Analysis

RNA Cloning

RNA Sequencing

Transcriptome Analysis

RNA Structure / Function

Ribosomal RNA Depletion

RNA Extraction for Real-Time PCR

Which RNA Isolation Kit is Right for Your qRT-PCR Experiment?

  Order now Order now Order now Order now
  Gold standard for highly pure, intact RNA High-quality RNA in less than 20 minutes High-throughput purification of RNA and DNA Complete, no purification system for qRT-PCR results
  Trizol Reagents PureLink Kits MagMAX Kits Cells to Ct Kits
Recommendations Most Cited, Top Seller     Best for qRT-PCR
Isolation method Organic reagent Silica filter, column format (convenience) Magnetic beads (scalability, flexibility) Chemical lysis (time to qRT-PCR results)
High throughput compatible No Yes (plate format available) Yes Yes
RT reagents included No No No Yes
qPCR reagents included No No No Yes
Prep time ~1hr < 20 minutes ~45 minutes 10 minutes
Compatible sample types Most sample types, particularly more difficult to lyse Cells and tissues Cells, blood, plants Cells
Amount of starting material 100 mg of tissue or 107 cells (requires 1ml reagent) Up to 200 mg tissue, 5x106 to 1 x 108 cells Up to 100 mg tissue, Up to 5x106 cells 1–10,000 cells

Eliminate Genomic DNA Contamination

Genomic DNA contamination can lead to false positive RT-PCR results. We offer a variety of Ambion tools for minimizing genomic DNA contamination from RNA samples prior to RT-PCR. For example, DNA-free DNase Treatment and Removal Reagents are designed for removing contaminating DNA from RNA samples and for the removal of DNase after treatment without Proteinase K treatment and organic extraction. In addition, we offer TURBO DNase enzyme kits, a hyperactive enzyme engineered from wild-type bovine DNase. The proficiency of TURBO DNase enzyme in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination. To prevent cross contamination during PCR experiments, we also offer DNAZap DNA Degradation Solution and RNase-free barrier pipette tips.

Technical Resources


Microarray Analysis

Advance your research with Affymetrix microarray analysis products

Thermo Fisher Scientific provides innovative Affymetrix products, tools, and resources that help advance the work of researchers via microarray analysis. Application areas that benefit from such an approach include plant and animal genomics and transcriptomics, including basic research and industrial application of technologies for breeding, population diversity and conservation, trait analysis, and more. Unique solutions are also available for cancer research from discovery to clinical research and validation, such as cytogenetics research of constitutional and cancer disorders. The genetics of human complex traits, Mendelian disorders, and populations are also application arrays that can be advanced with Affymetrix microarray analysis, such as through population-optimized and application-optimized genotyping to enable human genetic research workflows.

Featured products

Axiom 2.0 Reagent Kit

PharmacoScan Assay Kit

Clariom D Pico Assay

Microarray analysis categories

Microarrays are the platform of choice for detecting DNA structural variants (SV) such as chromosome insertions, duplications and deletions.

Microarray based assays for molecular cytogenetics research provide a genome-wide reliable approach that enables high-solution DNA copy number analysis.

The Axiom Genotyping Solution offers a uniquely flexible choice of customizable arrays or pre-designed arrays with imputation-aware designs covering more populations than any other technology.

Affymetrix agrigenomic genotyping solutions provide breeders and researchers with a powerful and flexible range of genotyping tools to cost-effectively identify, validate, and screen complex genetic traits in plants and animals.

Measuring the changes in the miRNA expression profile is extremely important for deciphering the biological context of differentially expressed genes.

Phenotypic abnormalities are rarely a result of expression changes in single genes, so generating a comprehensive expression profile is critical when studying normal biology and disease processes.

Instruments such as the GeneChip Scanner, as well as data analysis software and microarray analysis services

We provide a variety of tools, resources, analysis files, and sample data to support planning and execution of your experiment.

Get faster access to deeper data with the insight and efficient services of an Affymetrix partner.

Data management and end-to-end sample tracking for microarray workflows.

NetAffx analysis center

The NetAffx Analysis Center enables researchers to correlate their GeneChip array results with array design and annotation information.

  • Search probe sets for a term or identifier
  • Retrieve annotations for a probe list
  • Find probe sets that BLAST align to your sequence(s) through BLAST
  • Find probes that identically match your sequence(s)
  • Query the UCSC Browser including a custom track for your array of interest.

Visit the NetAffx Analysis Center 


RNA Sequencing Sample Preparation

Next-generation sequencing has transformed gene expression profiling and other RNA expression analysis studies. With an increasing number of researchers employing next-generation sequencing, and with the evolution of sequencing platforms, it is essential to use robust and streamlined methods for sample preparation. The best next-generation sequencing data begin with optimal samples.

Learn more about RNA & Transcriptome Sequencing

RNA Sequencing Overview

Next-generation sequencing studies require robust and streamlined methods for sample preparation. The best next-generation sequencing data begin with optimal samples. Click the tabs above to learn more and see all available products.

RNA Sequencing Overview

RNA Isolation

Although many methods are available for purifying RNA from any particular source, obtaining RNA of the highest purity is the best assurance that the downstream manipulations will be successful. Choose from various formats, including PureLink, mirVana, RecoverAll, and MagMAX kits to isolate total RNA, then enrich for the subpopulation of interest.

  Order now Order now Order now Order now Order now
  Simple, reliable, rapid method 30 min isolation from most samples HTP format, ideal for stringent applications Best for next-generation sequencing of mRNA High-throughput purification of FFPE samples
  PureLink RNA Mini Kit mirVana miRNA Isolation Kit (with or without phenol) MagMAX for Microarrays Total RNA Isolation Kit Dynabeads mRNA DIRECT Micro Kit MagMAX FFPE Total Nucleic Acid Isolation Kit
RNA types isolated for sequencing Large RNA molecules only (mRNA and rRNA) Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA) Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA) mRNA only total RNA, microRNA, gDNA
Isolation method Fast, convenient silica column Highest purity and convenience; includes organic extraction and silica column Scalable, flexible format for highest purity; includes organic extraction and magnetic beads Fast magnetic bead capture directly from microsized samples Xylene-free, scalable, flexible format with magnetic beads
Prep time 20 min 30 min <1 hr 15 min 3 hr (for 96 preps)
Amount of starting material 10 mg to 100 mg of tissue Up to 5 x 107 cells Up to 100 mg of tissue Up to 1 x 107 cells Up to 100 mg of tissue Up to 1 x 107 cells Up to 500 ng purified Total RNA 2 x 10 micron FFPE sections
Prep size 50 preps 40 preps 96 preps 2 mL oligo dT beads (sufficient for 100 mRNA preps) 96 preps

RNA Enrichment

To maximize the effectiveness of sequencing reagents, it is often necessary to enrich the sample for specific regions or targets of interest. This enrichment helps eliminate nonspecific artifacts, making it possible to better analyze smaller changes in genomes or transcriptomes and less abundant sequences. Ambion RiboMinus technology is designed to enrich the whole spectrum of RNA transcripts, regardless of their polyadenylation status or the presence of a 5' cap, by selectively depleting rRNA. The RiboMinus method removes the vast majority of rRNA (up to 99.9%) to allow greater interrogation of the less abundant transcripts.

Learn more about RiboMinus technology

  Order now Order now
  A novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNA A novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNA
  RiboMinus Eukaryote Kit for RNA-Seq RiboMinus Plant Kit for RNA-Seq
Platform Diagnostic Diagnostic
Starting material 1–10 µg total RNA 100–500 ng total RNA (low-input protocol) 1–10 µg total RNA
RiboMinus RNA yield 1–2 µg from 10 µg human total RNA ~100 ng from 500 ng human total RNA (low-input protocol) 1–2 µg from 10 µg human total RNA
Probe specificity Eukaryotic Plant
Cat. No. A1083708 - 8 preps A1083808 - 8 preps

RNA Library Construction

Library construction, template preparation, and sequencing are at the heart of the sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data. Generate cDNA by reverse transcription from adaptors ligated to the ends of RNA, then amplify the cDNA using primers complementary to the adaptors, and purify it.

  Order now Order now Order now
  Prepare representative cDNA libraries for strand-specific RNA sequencing A Great Solution for NGS The Ultimate Solution for NGS
  Ion Total RNA-Seq Kit v2 SOLiD Total RNA-Seq Kit Ambion RNA-Seq Library Construction Kit
Platform Ion Torrent PGM SOLiD 5500 Analyzer Illumina
RNA input type rRNA-depleted total RNA, poly(A) RNA, or miRNA Total RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNA Total RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNA
RNA input range Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome: 100 ng total RNA (~1 ng poly(A) or ~25 ng rRNA-RNA
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A), 200 ng
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A) or 200 ng rRNA-depleted total RNA, or 200 ng total RNA
Barcodes available Yes 96 No
Cat. No. 4475936 - 12 reaction kit 4445374 - 12 reaction kit 4454073 - 12 reaction kit

RNA Library Controls

Library construction, template preparation, and sequencing are at the heart of the sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data.

Learn more about ERCC Controls

  Order now Order now
  2 tubes containing preformulated mixes of the same 92 RNA transcripts but in different ratios, for measuring dynamic range, assessing bias, and confirming expression fold changes Single tube containing a preformulated mix of 92 RNA transcripts for measuring dynamic range and assessing bias
  ERCC ExFold RNA Spike-In Mixes (2 x 10 µL) ERCC RNA Spike-In Mix (1 x 10 µL)
Top Seller    
Compatible sample types Eukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA Eukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA
Dynamic range 6 logs 6 logs
Volume 10 µL (2 tubes) 10 µL
Number of reactions Spike-in controls diluted based on sample type and concentration Spike-in controls diluted based on sample type and concentration

RNA Barcoding

Sets of 16 barcodes are available for use with the SOLiD Total RNA-Seq Kit or the SOLiD SAGE Kit with Barcoding Adaptor Module. Barcodes cost-effectively increase the number of samples that can be analyzed in a single sequencing run. Barcodes assign a unique identifier to templated beads made from a single library. Once assigned, the RNA libraries can be pooled and sequenced.

RNA Sequencing Overview

Next-generation sequencing studies require robust and streamlined methods for sample preparation. The best next-generation sequencing data begin with optimal samples. Click the tabs above to learn more and see all available products.

RNA Sequencing Overview

RNA Isolation

Although many methods are available for purifying RNA from any particular source, obtaining RNA of the highest purity is the best assurance that the downstream manipulations will be successful. Choose from various formats, including PureLink, mirVana, RecoverAll, and MagMAX kits to isolate total RNA, then enrich for the subpopulation of interest.

  Order now Order now Order now Order now Order now
  Simple, reliable, rapid method 30 min isolation from most samples HTP format, ideal for stringent applications Best for next-generation sequencing of mRNA High-throughput purification of FFPE samples
  PureLink RNA Mini Kit mirVana miRNA Isolation Kit (with or without phenol) MagMAX for Microarrays Total RNA Isolation Kit Dynabeads mRNA DIRECT Micro Kit MagMAX FFPE Total Nucleic Acid Isolation Kit
RNA types isolated for sequencing Large RNA molecules only (mRNA and rRNA) Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA) Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA) mRNA only total RNA, microRNA, gDNA
Isolation method Fast, convenient silica column Highest purity and convenience; includes organic extraction and silica column Scalable, flexible format for highest purity; includes organic extraction and magnetic beads Fast magnetic bead capture directly from microsized samples Xylene-free, scalable, flexible format with magnetic beads
Prep time 20 min 30 min <1 hr 15 min 3 hr (for 96 preps)
Amount of starting material 10 mg to 100 mg of tissue Up to 5 x 107 cells Up to 100 mg of tissue Up to 1 x 107 cells Up to 100 mg of tissue Up to 1 x 107 cells Up to 500 ng purified Total RNA 2 x 10 micron FFPE sections
Prep size 50 preps 40 preps 96 preps 2 mL oligo dT beads (sufficient for 100 mRNA preps) 96 preps

RNA Enrichment

To maximize the effectiveness of sequencing reagents, it is often necessary to enrich the sample for specific regions or targets of interest. This enrichment helps eliminate nonspecific artifacts, making it possible to better analyze smaller changes in genomes or transcriptomes and less abundant sequences. Ambion RiboMinus technology is designed to enrich the whole spectrum of RNA transcripts, regardless of their polyadenylation status or the presence of a 5' cap, by selectively depleting rRNA. The RiboMinus method removes the vast majority of rRNA (up to 99.9%) to allow greater interrogation of the less abundant transcripts.

Learn more about RiboMinus technology

  Order now Order now
  A novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNA A novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNA
  RiboMinus Eukaryote Kit for RNA-Seq RiboMinus Plant Kit for RNA-Seq
Platform Diagnostic Diagnostic
Starting material 1–10 µg total RNA 100–500 ng total RNA (low-input protocol) 1–10 µg total RNA
RiboMinus RNA yield 1–2 µg from 10 µg human total RNA ~100 ng from 500 ng human total RNA (low-input protocol) 1–2 µg from 10 µg human total RNA
Probe specificity Eukaryotic Plant
Cat. No. A1083708 - 8 preps A1083808 - 8 preps

RNA Library Construction

Library construction, template preparation, and sequencing are at the heart of the sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data. Generate cDNA by reverse transcription from adaptors ligated to the ends of RNA, then amplify the cDNA using primers complementary to the adaptors, and purify it.

  Order now Order now Order now
  Prepare representative cDNA libraries for strand-specific RNA sequencing A Great Solution for NGS The Ultimate Solution for NGS
  Ion Total RNA-Seq Kit v2 SOLiD Total RNA-Seq Kit Ambion RNA-Seq Library Construction Kit
Platform Ion Torrent PGM SOLiD 5500 Analyzer Illumina
RNA input type rRNA-depleted total RNA, poly(A) RNA, or miRNA Total RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNA Total RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNA
RNA input range Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome: 100 ng total RNA (~1 ng poly(A) or ~25 ng rRNA-RNA
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A), 200 ng
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A) or 200 ng rRNA-depleted total RNA, or 200 ng total RNA
Barcodes available Yes 96 No
Cat. No. 4475936 - 12 reaction kit 4445374 - 12 reaction kit 4454073 - 12 reaction kit

RNA Library Controls

Library construction, template preparation, and sequencing are at the heart of the sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data.

Learn more about ERCC Controls

  Order now Order now
  2 tubes containing preformulated mixes of the same 92 RNA transcripts but in different ratios, for measuring dynamic range, assessing bias, and confirming expression fold changes Single tube containing a preformulated mix of 92 RNA transcripts for measuring dynamic range and assessing bias
  ERCC ExFold RNA Spike-In Mixes (2 x 10 µL) ERCC RNA Spike-In Mix (1 x 10 µL)
Top Seller    
Compatible sample types Eukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA Eukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA
Dynamic range 6 logs 6 logs
Volume 10 µL (2 tubes) 10 µL
Number of reactions Spike-in controls diluted based on sample type and concentration Spike-in controls diluted based on sample type and concentration

RNA Barcoding

Sets of 16 barcodes are available for use with the SOLiD Total RNA-Seq Kit or the SOLiD SAGE Kit with Barcoding Adaptor Module. Barcodes cost-effectively increase the number of samples that can be analyzed in a single sequencing run. Barcodes assign a unique identifier to templated beads made from a single library. Once assigned, the RNA libraries can be pooled and sequenced.

RNA Sequencing

No matter which platform you choose to suit your needs, we continue to support the evolving needs of this field with gold-standard instrumentation, innovative next-generation technology, optimized best-in-class reagents, consumables, analysis software, applications and world-class technical support.

Next-Generation (SOLiD) 
Sequence right here, right now—minimizing wait time and costs for wasted reagents.

Capillary Electrophoresis Sequencing
The gold standard in sequencing and a fast, flexible platform for analysis.

Semiconductor (Ion Torrent) Sequencing
Sequence from your benchtop with Ion Torrent revolutionary "on-chip" technology.

RNA & Transcriptome Sequencing
Choose the correct technology for your specific RNA application.

Latest Technical Resources

Publications & Literature

Webinars


Northern Blot Analysis

Northern blot analysis reveals information about RNA identity, size, and abundance, allowing a deeper understanding of gene expression levels. Our Invitrogen portfolio comprises one of the industry’s most comprehensive product offerings for northern blot analysis. From Invitrogen TRIzol reagent that enables high-quality, intact RNA preparations to RNase-free reagents and Invitrogen BrightStar-Plus membranes, this array of products meets all your Northern blot analysis needs and helps shorten the time it takes you to get results.

Step-by-Step Guide to Northern Blot Analysis

RNA Isolation

Obtaining high-quality, intact RNA is a critical step in performing northern blot analysis. All protocols, techniques, and commercially available kits used to isolate RNA share these common attributes:

  • Cellular lysis and membrane disruption
  • Inhibition of ribonuclease activity
  • Deproteinization
  • Recovery of intact RNA

Products for RNA Isolation

IS10008 iPrep PureLink Virus Kit K156001 PureLink Total RNA Blood Kit
AM1934 LeukoLOCK Total RNA Isolation System 12280050 PureLink Viral RNA/DNA Mini Kit
AM1840 MagMAX Total Nucleic Acid Isolation Kit AM1975 RecoverAll Total Nucleic Acid Isolation Kit for FFPE
AM1839 MagMAX-96 for Microarrays Total RNA Isolation Kit AM1925 RiboPure-Bacteria/Yeast Kit
AM1830 MagMAX-96 Total RNA Isolation Kit AM1926 RiboPure-Yeast Kit
AM1936 MagMAX-96 Viral RNA Isolation Kit  AM1928 RiboPure-Blood Kit
AM1983 MELT Total Nucleic Acid Isolation System AM1912 RNAqueous Kit
AM9690 Plant RNA Isolation Aid  AM1931 RNAqueous-Micro Kit
12322012 Plant RNA Reagent 4380204 Tempus Spin RNA Isolation Kit
12173011A PureLink Pro 96 Total RNA Purification Kit 10296010 TRIzol LS Reagent
12280096A PureLink Pro 96 Viral RNA/DNA Purification Kit 16096020 TRIzol Max Bacteria/Yeastl RNA Isolation Kit
12183016 PureLink RNA Micro Kit 12183555 TRIzol Plus RNA Purification System
12183018A PureLink RNA Mini Kit 15596026 TRIzol Reagent

View our RNA Isolation Kit Selection Guide

Probe Generation

RNA probes can be produced by in vitro transcription reactions using the Invitrogen MAXIscript Kit. Isotope- or nonisotope-labeled nucleotides can be incorporated directly during synthesis with this kit, or the RNA can be synthesized unlabeled and subsequently treated with Psoralen-Biotin to produce biotinylated probes for blot hybridizations.

Technical Considerations:

When performing northern blot analyses using RNA probes rather than DNA probes, we recommend that several adjustments be made to obtain the best results. These include:

  • The type of membrane used
  • RNA-appropriate hybridization buffer
  • Elevated hybridization and wash temperatures
  • Probe techniques.

Products for Probe Generation

AM1308M MAXIscript SP6 Kit with Manual
AM1320M MAXIscript SP6⁄T7 Kit (15 rxns each) with Manual
AM1316M MAXIscript T3 Kit with Manual
AM1312M MAXIscript T7 Kit with Manual
AM1324M MAXIscript T7⁄T3 Kit (15 rxns each) with Manual

Denaturing

Once RNA samples are isolated, the next step in northern blot analysis is denaturing agarose gel electrophoresis. Formaldehyde has been the denaturant traditionally used during electrophoresis. The Invitrogen NorthernMax kit contains a complete set of RNase-free reagents for running formaldehyde-based agarose gels. The disadvantage of using formaldehyde is the need to pour and run gels in a fume hood. With the Invitrogen NorthernMax-Gly Kit, RNA samples are denatured in glyoxal/DMSO and run without the safety issues associated with formaldehyde.

A wide range of RNA ladders are available for accurate mRNA size and mass estimations, including the Invitrogen Millennium RNA Markers. Go to RNA ladders for more information.

Products for Denaturing

AM7785 Millennium Marker Probe Template 750018 50x Denhardt’s solution
AM8676 NorthernMax 10X Denaturing Gel Buffer 750023 UltraPure DEPC-treated water
AM8552 NorthernMax Formaldehyde Load Dye (1 mL tubes) 10977015 UltraPure DNAse/RNase Free Distilled water
AM1940 NorthernMax Kit 16550100 UltraPure Agarose 1000
AM8672 NorthernMax One-Hour Transfer Buffer 14476028 UltraPure 0.5M EDTA, pH 8.0
AM8677 NorthernMax Prehybridization⁄ Hybridization Buffer 24740011 UltraPure 5M NaCl
AM8678 NorthernMax-Gly 10X Gel Prep⁄Running Buffer 15506017 UltraPure Tris HCl
AM1946 NorthernMax-Gly Kit 15557036 UltraPure 20X SSC
AM8551 NorthernMax-Gly Sample Loading Dye (1 mL tubes) 15591035 UltraPure 20X SSPE

Transfer to Solid Support and Immobilization

Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and immobilized for subsequent hybridization. For fast, reproducible transfer, the iBlot Dry Blotting System offers complete transfer of RNA to nylon membrane typically in 7 minutes. With dry blotting, there is no need for additional buffer or liquids that can introduce variability into the result.

Alternatively, we offer a rapid alkaline transfer method that is incorporated into the NorthernMax and NorthernMax-Gly procedure. Instead of overnight transfer, the transfer takes about 2 hours and delivers higher blot sensitivity by efficiently moving RNA (especially larger transcripts) onto the membrane. The NorthernMax and NorthernMax-Gly Kits have been optimized to work with the BrightStar-Plus positively charged nylon membranes, and we recommend their use to minimize background and maximize signal. Please note that nitrocellulose membranes are chemically incompatible with the NorthernMax Transfer Buffer and should not be used with these kits.

Products for Transferring to Solid Support and Immobilization

AM10100 BrightStar-Plus Positively Charged Nylon Membrane (15 x 15 cm)
AM10104 BrightStar-Plus Positively Charged Nylon Membrane (large roll, 30 cm x 3 m)
AM10102 BrightStar-Plus Positively Charged Nylon Membrane (small roll, 30 x 45 cm)
15515026 UltraPure Formamide
AM9342 Formamide (Deionized)
LC2003 Novex Nylon Positively Charged Membrane, 045 micron (8.3 x 7.3 cm)

Prehybridization and Hybridization with Probe

The Invitrogen NorthernMax and NorthernMax-Gly kits include ULTRAhyb Ultrasensitive Hybridization Buffer, which can be used for both prehybridization and hybridization. Although double-stranded DNA probes must be denatured prior to use, RNA probes and single-stranded DNA probes can be diluted in a small amount of ULTRAhyb buffer and then added to the prehybridized blot. ULTRAhyb buffer can increase sensitivity up to 100-fold compared to other hybridization solutions by pushing hybridization to completion without increasing background. As few as 10,000 molecules can be detected. Because ULTRAhyb buffer maximizes blot sensitivity, typically hybridization can be performed in just 2 hours for many messages.

Products for Prehybridization and Hybridization with Probe

AM8676 NorthernMax 10X Denaturing Gel Buffer
AM8552 NorthernMax Formaldehyde Load Dye (1 ml tubes)
AM1940 NorthernMax Kit
AM8672 NorthernMax One-Hour Transfer Buffer
AM8677 NorthernMax Prehybridization⁄ Hybridization Buffer
AM8678 NorthernMax-Gly 10X Gel Prep⁄Running Buffer
AM1946 NorthernMax-Gly Kit
AM8551 NorthernMax-Gly Sample Loading Dye (1 ml tubes)

Washing

After hybridization, unhybridized probe is removed by washing in several changes of buffer. Low stringency washes (e.g., with 2X SSC or SSPE) remove the hybridization solution and unhybridized probe. High stringency washes (e.g., with 0.1X SSC or SSPE) remove partially hybridized molecules. Certified RNase-free low- and high-stringency wash buffers are included in the NorthernMax Kits, and are also available separately.

Products for Washing

AM8677 NorthernMax Prehybridization/ Hybridization Buffer
AM8670 ULTRAhyb Ultrasensitive Hybridization Buffer
AM8669 ULTRAhyb Ultrasensitive Hybridization Buffer
AM8663 ULTRAhyb-Oligo

Detection

The BrightStar BioDetect Nonisotopic Detection Kit provides all the reagents and materials necessary for detection of biotinylated RNA and DNA probes. The NorthernMax Kits have been optimized in conjunction with the BrightStar BioDetect Kit for ultrasensitive nonisotopic northern blots with a high signal-to-noise ratio and low background. This optimization of the northern blotting method also yields excellent results with radiolabeled probes.

Products for Detection

AM1930 BrightStar BioDetect Kit

RNA Isolation

Obtaining high-quality, intact RNA is a critical step in performing northern blot analysis. All protocols, techniques, and commercially available kits used to isolate RNA share these common attributes:

  • Cellular lysis and membrane disruption
  • Inhibition of ribonuclease activity
  • Deproteinization
  • Recovery of intact RNA

Products for RNA Isolation

IS10008 iPrep PureLink Virus Kit K156001 PureLink Total RNA Blood Kit
AM1934 LeukoLOCK Total RNA Isolation System 12280050 PureLink Viral RNA/DNA Mini Kit
AM1840 MagMAX Total Nucleic Acid Isolation Kit AM1975 RecoverAll Total Nucleic Acid Isolation Kit for FFPE
AM1839 MagMAX-96 for Microarrays Total RNA Isolation Kit AM1925 RiboPure-Bacteria/Yeast Kit
AM1830 MagMAX-96 Total RNA Isolation Kit AM1926 RiboPure-Yeast Kit
AM1936 MagMAX-96 Viral RNA Isolation Kit  AM1928 RiboPure-Blood Kit
AM1983 MELT Total Nucleic Acid Isolation System AM1912 RNAqueous Kit
AM9690 Plant RNA Isolation Aid  AM1931 RNAqueous-Micro Kit
12322012 Plant RNA Reagent 4380204 Tempus Spin RNA Isolation Kit
12173011A PureLink Pro 96 Total RNA Purification Kit 10296010 TRIzol LS Reagent
12280096A PureLink Pro 96 Viral RNA/DNA Purification Kit 16096020 TRIzol Max Bacteria/Yeastl RNA Isolation Kit
12183016 PureLink RNA Micro Kit 12183555 TRIzol Plus RNA Purification System
12183018A PureLink RNA Mini Kit 15596026 TRIzol Reagent

View our RNA Isolation Kit Selection Guide

Probe Generation

RNA probes can be produced by in vitro transcription reactions using the Invitrogen MAXIscript Kit. Isotope- or nonisotope-labeled nucleotides can be incorporated directly during synthesis with this kit, or the RNA can be synthesized unlabeled and subsequently treated with Psoralen-Biotin to produce biotinylated probes for blot hybridizations.

Technical Considerations:

When performing northern blot analyses using RNA probes rather than DNA probes, we recommend that several adjustments be made to obtain the best results. These include:

  • The type of membrane used
  • RNA-appropriate hybridization buffer
  • Elevated hybridization and wash temperatures
  • Probe techniques.

Products for Probe Generation

AM1308M MAXIscript SP6 Kit with Manual
AM1320M MAXIscript SP6⁄T7 Kit (15 rxns each) with Manual
AM1316M MAXIscript T3 Kit with Manual
AM1312M MAXIscript T7 Kit with Manual
AM1324M MAXIscript T7⁄T3 Kit (15 rxns each) with Manual

Denaturing

Once RNA samples are isolated, the next step in northern blot analysis is denaturing agarose gel electrophoresis. Formaldehyde has been the denaturant traditionally used during electrophoresis. The Invitrogen NorthernMax kit contains a complete set of RNase-free reagents for running formaldehyde-based agarose gels. The disadvantage of using formaldehyde is the need to pour and run gels in a fume hood. With the Invitrogen NorthernMax-Gly Kit, RNA samples are denatured in glyoxal/DMSO and run without the safety issues associated with formaldehyde.

A wide range of RNA ladders are available for accurate mRNA size and mass estimations, including the Invitrogen Millennium RNA Markers. Go to RNA ladders for more information.

Products for Denaturing

AM7785 Millennium Marker Probe Template 750018 50x Denhardt’s solution
AM8676 NorthernMax 10X Denaturing Gel Buffer 750023 UltraPure DEPC-treated water
AM8552 NorthernMax Formaldehyde Load Dye (1 mL tubes) 10977015 UltraPure DNAse/RNase Free Distilled water
AM1940 NorthernMax Kit 16550100 UltraPure Agarose 1000
AM8672 NorthernMax One-Hour Transfer Buffer 14476028 UltraPure 0.5M EDTA, pH 8.0
AM8677 NorthernMax Prehybridization⁄ Hybridization Buffer 24740011 UltraPure 5M NaCl
AM8678 NorthernMax-Gly 10X Gel Prep⁄Running Buffer 15506017 UltraPure Tris HCl
AM1946 NorthernMax-Gly Kit 15557036 UltraPure 20X SSC
AM8551 NorthernMax-Gly Sample Loading Dye (1 mL tubes) 15591035 UltraPure 20X SSPE

Transfer to Solid Support and Immobilization

Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and immobilized for subsequent hybridization. For fast, reproducible transfer, the iBlot Dry Blotting System offers complete transfer of RNA to nylon membrane typically in 7 minutes. With dry blotting, there is no need for additional buffer or liquids that can introduce variability into the result.

Alternatively, we offer a rapid alkaline transfer method that is incorporated into the NorthernMax and NorthernMax-Gly procedure. Instead of overnight transfer, the transfer takes about 2 hours and delivers higher blot sensitivity by efficiently moving RNA (especially larger transcripts) onto the membrane. The NorthernMax and NorthernMax-Gly Kits have been optimized to work with the BrightStar-Plus positively charged nylon membranes, and we recommend their use to minimize background and maximize signal. Please note that nitrocellulose membranes are chemically incompatible with the NorthernMax Transfer Buffer and should not be used with these kits.

Products for Transferring to Solid Support and Immobilization

AM10100 BrightStar-Plus Positively Charged Nylon Membrane (15 x 15 cm)
AM10104 BrightStar-Plus Positively Charged Nylon Membrane (large roll, 30 cm x 3 m)
AM10102 BrightStar-Plus Positively Charged Nylon Membrane (small roll, 30 x 45 cm)
15515026 UltraPure Formamide
AM9342 Formamide (Deionized)
LC2003 Novex Nylon Positively Charged Membrane, 045 micron (8.3 x 7.3 cm)

Prehybridization and Hybridization with Probe

The Invitrogen NorthernMax and NorthernMax-Gly kits include ULTRAhyb Ultrasensitive Hybridization Buffer, which can be used for both prehybridization and hybridization. Although double-stranded DNA probes must be denatured prior to use, RNA probes and single-stranded DNA probes can be diluted in a small amount of ULTRAhyb buffer and then added to the prehybridized blot. ULTRAhyb buffer can increase sensitivity up to 100-fold compared to other hybridization solutions by pushing hybridization to completion without increasing background. As few as 10,000 molecules can be detected. Because ULTRAhyb buffer maximizes blot sensitivity, typically hybridization can be performed in just 2 hours for many messages.

Products for Prehybridization and Hybridization with Probe

AM8676 NorthernMax 10X Denaturing Gel Buffer
AM8552 NorthernMax Formaldehyde Load Dye (1 ml tubes)
AM1940 NorthernMax Kit
AM8672 NorthernMax One-Hour Transfer Buffer
AM8677 NorthernMax Prehybridization⁄ Hybridization Buffer
AM8678 NorthernMax-Gly 10X Gel Prep⁄Running Buffer
AM1946 NorthernMax-Gly Kit
AM8551 NorthernMax-Gly Sample Loading Dye (1 ml tubes)

Washing

After hybridization, unhybridized probe is removed by washing in several changes of buffer. Low stringency washes (e.g., with 2X SSC or SSPE) remove the hybridization solution and unhybridized probe. High stringency washes (e.g., with 0.1X SSC or SSPE) remove partially hybridized molecules. Certified RNase-free low- and high-stringency wash buffers are included in the NorthernMax Kits, and are also available separately.

Products for Washing

AM8677 NorthernMax Prehybridization/ Hybridization Buffer
AM8670 ULTRAhyb Ultrasensitive Hybridization Buffer
AM8669 ULTRAhyb Ultrasensitive Hybridization Buffer
AM8663 ULTRAhyb-Oligo

Detection

The BrightStar BioDetect Nonisotopic Detection Kit provides all the reagents and materials necessary for detection of biotinylated RNA and DNA probes. The NorthernMax Kits have been optimized in conjunction with the BrightStar BioDetect Kit for ultrasensitive nonisotopic northern blots with a high signal-to-noise ratio and low background. This optimization of the northern blotting method also yields excellent results with radiolabeled probes.

Products for Detection

AM1930 BrightStar BioDetect Kit

Dry, Bufferless Transfer

iBlot Dry Blotting System

iBlot Dry Blotting System

  • Time savings—transfer DNA or RNA typically in 7 minutes or less
  • Reproducibility—consistent preparation and running conditions
  • Convenience—no messy buffers or laborious preparation

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RNA Cloning

Obtaining high-quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments, including RNA cloning. For RNA cloning, we recommend starting with polyadenylated RNA (poly(A)+ RNA), which comprises messenger RNA (mRNA) rather than total RNA. Our Dynabeads mRNA DIRECT products employ a magnetic capture-bead strategy to offer maximum RNA yield, purity, and integrity from a wide range of sample types.

Dynabeads mRNA DIRECT Kit

  • Designed for a broad range of sample types, including blood, serum, and plasma; mammalian, amphibian, fish, plant, and insect tissues; FFPE samples; and yeast
  • Physical mRNA capture on mobile magnetic beads for maximum poly(A)+ RNA recovery
  • Recovered mRNA is suitable for virtually any downstream application, including RNA cloning, RACE, cDNA library construction, quantitative RT-PCR, SAGE, ribonuclease protection assays, subtractive hybridization, and primer extension
  • Versatile elution options, including eluting in as little as 5 µL or skipping elution and adding Dynabeads magnetic beads to your downstream reaction

Cloning Workflow

Ordering Information

Resources


In Vitro Transcription & Translation

In vitro RNA transcription reactions are generally used for two distinct purposes: the synthesis of labeled probes, and the synthesis of large amounts of unlabeled RNA. Additionally, capped RNA synthesized in transcription reactions is used for microinjection, in vitro translation, and transfection. The RNA experts at Life Technologies have developed nucleotides, polymerases, modifying enzymes, and kits for high-yield capped RNA transcription, large-scale transcription, and transcription of short DNA templates. We also offer a complete line of Ambion products for in vitro transcription and translation studies.

Tools for In Vitro Transcription & Translation

In Vitro Transcription

The ability to synthesize RNA in the laboratory is critical to many techniques. Radiolabeled and nonisotopically labeled RNA probes, generated in small-scale transcription reactions, can be used in blot hybridizations and nuclease protection assays. mRNA amplification for gene array analysis requires the use of large-scale transcription reactions.

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In Vitro Translation

In vitro synthesis of proteins in cell-free extracts is an important tool for molecular biologists and has a variety of applications, including the rapid identification of gene products (e.g., proteomics), localization of mutations through synthesis of truncated gene products, protein folding studies, and incorporation of modified or unnatural amino acids for functional studies.

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Technical Resources


Transcriptome Analysis

Whole-transcriptome analysis is of growing importance in understanding how altered expression of genetic variants contributes to complex diseases such as cancer, diabetes, and heart disease. Analysis of genome-wide differential RNA expression provides researchers with greater insights into biological pathways and molecular mechanisms that regulate cell fate, development, and disease progression. We offer an extensive range of products, from microarray labeling reagents to next-generation sequencing reagents and instrumentation, to help capture the complexity of whole-transcriptome analysis for your research.

RNA Purification & Transcriptome Enrichment

Successful whole transcriptome analysis depends on RNA quality and efficient conversion to cDNA libraries. Invitrogen and Ambion Transcriptome analysis sample preparation solutions are designed to be optimized for your desired workflow. Optimized sample preparation means complete solutions from one source, that minimize further manipulation and are flexible enough to work with a broad range of starting samples, for greater confidence, and accountability to help troubleshoot when needed.

Expression Microarrays Labeling

Gene arrays have become a powerful approach for comparing complex sample RNA populations. Using array analysis, the expression profiles of normal and tumor tissues, treated and untreated cell cultures, developmental stages of an organism or tissue, and different tissues can be compared to gain a better understanding of the Transcriptome.

Next-Generation Sequencing

With a dynamic range to detect subtle changes in expression level in a hypothesis-neutral environment, next-generation sequencing helps provide an understanding of biological response to stimuli or environmental changes. The Ion Proton System offers a reliable sequencing workflow for your research that combines simple sample preparation and intuitive data analysis with the flexibility to perform whole-transcriptome sequencing for identification and quantification of novel and known transcripts, or targeted transcriptome sequencing for simple, gene-level expression analysis.


RNA Structure / Function

RNA structure is thought to play a central role in many cellular processes, including transcription initiation, elongation and termination, mRNA splicing, and retroviral infection of eukaryotic cells. Elucidating the mechanistic aspects of these intricate processes will require detailed understanding of the underlying RNA structure. RNA molecules usually possess a variety of single-stranded and double-stranded regions that give rise to complex three-dimensional structures. These structures are involved in the molecule's interactions with other nucleic acids, proteins, and small molecules. We have developed an extensive portfolio of products for the synthesis and modification of RNA in order to further understand the role of RNA structure.

Enzymes

  • RNA-Grade Ribonucleases: A collection of ribonucleases (RNases A and T1) that are optimized for researchers performing RNA structure, RNA sequencing, protein footprinting and boundary experiments.
  • Poly(A)Polymerase: Catalyzes the addition of adenosine to the 3' end of RNA in a sequence-independent fashion.
  • T7 RNA Polymerase: Catalyzes the 5´→3´ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter.
  • SP6 RNA Polymerase: Catalyzes the 5´→3´ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter and incorporates modified nucleotides.
  • Anza T4 Polynucleotide Kinase: Transfers the terminal phosphate of ATP to the 5´ hydroxyl terminus of DNA or RNA. Used for 5' end labeling of oligonucleotides and polynucleotides.
  • T4 RNA Ligase: Catalyzes the formation of a phosphodiester linkage between a 5´-phosphoryl-terminated ribonucleic acid and a 3´-hydroxyl-terminated ribonucleic acid.
  • Terminal Transferase: Catalyzes the addition of deoxynucleotides to the 3´ hydroxyl terminus of DNA.

In Vitro transcription kits

  • MEGAscript SP6T7 and T3 Kits: Synthesize ultra-high yields of RNA, 10 to 50 times as much as produced by conventional transcription reactions.

Other

  • Cap Analog & Variants: m7G(5´)ppp(5´)G (Cap Analog) and Cap Variants. The Cap Analog is used for synthesis of 5´-capped RNA molecules.
  • Modified UTPs: Incorporate these nucleotides to confer unique characteristics and test for particular reactive groups.

References

  1. Moine H, Ehresmann B, Ehresmann C, and Romby P. (1998) Probing RNA structure and function in solution. In: Simons RW and Grunberg-Manago M (editors). RNA Structure and Function, Cold Spring Harbor Laboratory Press. p. 77-115. 
  2. Knapp G. (1989) Enzymatic approaches to probing of RNA secondary and tertiary structure. Methods Enzymol 180:192-212. Abstract
  3. Krol A and Carbon P. (1989) A guide for probing native small nuclear RNA and ribonucleoprotein structures. Methods Enzymol 180:212-227. Abstract
  4. Tinoco I Jr, and Bustamante C. (1999) How RNA folds. J Mol Biol 293(2):271-281. Abstract
  5. Doudna J A. (2000) Structural genomics of RNA. Nature Struct Biol 7 Suppl:954-956. Abstract

Ribosomal RNA Depletion

Our understanding of transcriptome biology is undergoing a revolution that is revealing that the regulation of expression and the types and functions of RNA are far more complex than was previously thought. Enrichment of whole transcriptome RNA by depleting ribosomal RNA (rRNA) species using our RiboMinus technology has the potential to enhance discovery using gene expression microarrays, RNA-Seq, and other methods. The Ambion WT Expression Kit for RNA amplification prior to microarray analysis circumvents the need to deplete rRNA by selectively eliminating rRNA from reverse transcription in the amplification process. Both of these technologies have the potential to facilitate our understanding of the new paradigm forming around the role of the transcriptome in both normal physiological and pathological processes.

RiboMinus Technology

Our RiboMinus technology utilizes specific locked nucleic acid (LNA) capture probes to bind ribosomal RNA and subsequently remove it from the sample via binding to streptavidin-coated Dynabeads magnetic beads. The remaining whole-transcriptome RiboMinus RNA is suitable for direct sequencing using any next-generation sequencing platforms or microarray analysis.

Learn more about RiboMinus Technology

Ribosomal Binding Site Sequence Requirements

The sequence and structure of the 5' untranslated region (UTR) of mRNA transcripts plays a significant role in regulation of protein synthesis. In prokaryotes, the ribosome binding site (RBS), which promotes efficient and accurate translation of mRNA, is called the Shine-Dalgarno sequence after the scientists who first described it. This purine-rich 5' UTR sequence is complementary to the UCCU core sequence of the 3'-end of 16S rRNA (located within the 30S small ribosomal subunit). Various Shine-Dalgarno sequences have been found in prokaryotic mRNAs (see Figure 1 for the consensus sequence). These sequences lie about 10 nucleotides upstream from the AUG start codon. Activity of prokaryotic RBSs can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG.

Consensus RBS sequences

Figure 1. Consensus RBS Sequences. The +1 A is the first base of the AUG initiator codon (shaded) responsible for binding of fMet-tRNAfMet. The underline indicates the ribosomal binding site sequence, which is required for efficient translation

In eukaryotes, the Kozak sequence, A/GCCACCAUGG, which lies within a short 5' UTR, helps direct translation of mRNA. mRNAs lacking the Kozak consensus sequence may be translated efficiently in in vitro translation systems if they possesses a moderately long 5' UTR that lacks stable secondary structure. Our data demonstrate that in contrast to the E. coli ribosome, which preferentially recognizes the Shine-Dalgarno sequence, eukaryotic ribosomes (such as those found in retic lysate used in in vitro translation systems) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites.

Ribosomal RNA Sizes

Species rRNA Size (kb)
Human 18S  1.9
28S  5
Mouse 18S  1.9
28S  4.7
Drosophila 18S  2
28S  4.1*
Tobacco Leaf 16S  1.5
18S  1.9
23S  2.9
25S  3.7
Yeast 18S  2
26S  3.8
E. coli 16S  1.5
23S  2.9
Xenopus 18S  4
28S  1.8

*Drosophila 28S ribosomal RNA is processed into 2 fragments that migrate in a similar manner to the 18S rRNA.

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