Log In Sign up
The largest database of trusted experimental protocols
> Devices > Research Device > Northern Blot

Northern Blot

Northern blot is a widely used technique in molecular biology for the detection and quantification of specific RNA molecules within a total RNA sample.
This analytical method involves the separation of RNA molecules by size through gel electrophoresis, transfer to a membrane, and subsequent detection using labeled complementary DNA or RNA probes.
Northern blot analysis provides important insights into gene expression, allowing researchers to study the abundance and size of target RNAs.
PubCompare.ai, an AI-driven platform, can enhance the reproducibilty and accuracy of your Northern blot experiments by helping you easily locate protocols from literature, pre-prints, and patents, and leveraging AI-driven comparisons to identify the best protocols and products.
Optimize your Northern blot workflow with PubCompare.ai's powerful tools and experience the difference in your reseach today!

Most cited protocols related to «Northern Blot»

1
RNA Extraction and Northern Blot Analysis
Total RNA was extracted from virus-infected plants using the Direct-zol RNA MiniPrep Plus (Zymo Research) according to the manufacturer’s recommendations. For each sample, 10–15 μg of RNA was separated on a denaturing 2% agarose gel, blotted on a Hybond-N+ (GE Healthcare) membrane and hybridized with a DIG-labelled probe. For virus expression analysis, a DIG-labelled RNA probe was synthesized using DIG Northern Starter Kit (Roche) and manufacturer’s instructions were followed. For crRNA detection, a 5’-end DIG-labelled oligonucleotide (IDT) was used. DIG application manual (Roche) was followed for capillary transfer, hybridization, and detection. Northern blots were repeated in three independent experiments with the same results.
Aman R., Ali Z., Butt H., Mahas A., Aljedaani F., Khan M.Z., Ding S, & Mahfouz M. (2018). RNA virus interference via CRISPR/Cas13a system in plants. Genome Biology, 19, 1.
Full text: Click here
Publication 2018
Capillaries Crossbreeding Northern Blot Oligonucleotides Plant Viruses RNA, CRISPR Guide RNA Probes Sepharose Tissue, Membrane Virus
2
Evaluation of circRNA Abundance
RT-PCR and Northern blots were used to evaluate the relative abundance of circRNAs as described (Zhang et al. 2013 (link), 2014 (link)). PCR bands with novel circRNA-predominant exons were further subjected to Sanger sequencing. PCR primers and Northern blot probes are listed in Supplemental Table S6.
Zhang X.O., Dong R., Zhang Y., Zhang J.L., Luo Z., Zhang J., Chen L.L, & Yang L. (2016). Diverse alternative back-splicing and alternative splicing landscape of circular RNAs. Genome Research, 26(9), 1277-1287.
Full text: Click here
Publication 2016
Exons Northern Blot Oligonucleotide Primers Reverse Transcriptase Polymerase Chain Reaction RNA, Circular
3
Quantification of L1 RNA Abundance
Total RNA was isolated from transfected cell lys using TRIzol-LS (Invitrogen) and quantified (Nanodrop, Thermo Scientific). For qRTPCR analysis of transcript abundance, RNA (2 μg) was treated with RQ1 DNase (Promega) and cDNA synthesized from 1 µg using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). 2 µl cDNA were used for quantitative PCR using a TaqMan probe assay as described (5 (link)). For northern blots, total RNA and oligo dT-selected (poly A+, PureBiotech, MPG mRNA purification kit) RNA was fractionated through agarose formaldehyde gels, transferred to membrane and hybridized to 32P-labeled in vitro transcribed antisense L1 RNA (NorthernMax Kit, Ambion). Phosphorimages were captured on a Typhoon 9400 and analysed with ImageQuant 5.2 (GE Healthcare). For RT-PCR to detect the AI, cDNA was synthesized using random hexamer from either 500 ng polyA+ or 2µg total RNA template in a 20 µl reaction, then 1 µl of that was amplified in a 25 µl PCR reaction using GFPint1F, 5′-GACTGGGTGCTCAGGTAGTGG and GFPint1R 5′-AAGATCCGCCACAACATCGA for 40 cycles. Products were separated on 2% agarose, stained with SYBRSafe (Invitrogen), and imaged on the Typhoon9400.
Peddigari S., Li P.W., Rabe J.L, & Martin S.L. (2012). hnRNPL and nucleolin bind LINE-1 RNA and function as host factors to modulate retrotransposition. Nucleic Acids Research, 41(1), 575-585.
Publication 2012
Antisense RNA Biological Assay Cells Deoxyribonucleases DNA, Complementary Formaldehyde Gels Northern Blot oligo (dT) Poly A Promega Reverse Transcriptase Polymerase Chain Reaction Reverse Transcription RNA, Messenger Sepharose Tissue, Membrane trizol Typhoons
4
3' RACE and Alternative Splicing Analysis

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2009
Cold Temperature DNA, Complementary Genes Nested Genes Nested Polymerase Chain Reaction Northern Blot Oligonucleotide Primers Protein Isoforms RNA, Messenger RNA-Directed DNA Polymerase Sepharose
5
Comprehensive Plant RNA Extraction and Analysis
For isolating total RNA from plant samples, 1 ml of TRIzol per 0.2 g of plant tissue was used for extraction, and phenol:chloroform:isoamyl alcohol and chloroform were added for phase separation, followed by ethanol precipitation.
Nothern blotting was performed as described previously by Wu et al.19 (link). Briefly, 30 µg of total RNA was loaded onto a 19% PAGE gel for electrophoresis at 150 V for 4 h, after which the gels were transferred to a Hybond membrane (Amersham Biosciences, GE Healthcare) subjected to 200 mA for 2 h. After UV cross-linking was performed, the membrane was then hybridized in ULTRAhyb® Ultrasensitive Hybridizaton Buffer (Ambion, Austin, TX, USA) with DNA oligo probes. The probes were in the antisense orientation to the mature miRNA or U6 transcripts, with biotin labels at their 3′ terminal (TaKaRa, Otsu, Japan). The northern blot results were generated with a Light Shift EMSA Kit (Thermo Scientific, Waltham, MA, USA) and imaged using a FLA-5000 Phosphor imager (Fujifilm).
Total RNA samples were extracted from plant leaves using TRIzol, extracted with phenol:chloroform:isoamyl alcohol and chloroform, and then precipitated with ethanol.
For northern blotting, 30 µg of total RNA was resolved by 19% PAGE electrophoresis in 1 × TBE buffer and then transferred to a Hybond membrane (Amersham Biosciences, GE Healthcare), which was subjected to 200 mA for 2 h. The UV cross-linked membrane was subsequently hybridized in ULTRAhyb® Ultrasensitive Hybridizaton Buffer (Ambion, Austin, TX, USA) using antisense probes of 3′-biotin-labeled oligo DNA (TaKaRa, Otsu, Japan) to mature miRNA or U6 transcripts. The hybridization signals were developed with a Light Shift EMSA Kit (Thermo Scientific, Waltham, MA, USA) and imaged using a FLA-5000 Phosphor imager (Fujifilm).
To perform quantitative real-time PCR, 50 μg of RNA was treated with DNase I (TaKaRa) to remove DNA contamination, followed by RNA extraction with phenol:chloroform. Five micrograms of RNA was reverse transcribed to produce cDNAs with PrimeScript® Reverse Transcriptase (TaKaRa) in conjunction with oligo(dT) primers. Real-time PCR was performed with specific primer pairs (Supplementary Table 5) in a MyiQ2 Two-color Real-time PCR Detection System (Bio-Rad, Richmond, CA, USA). At least 3 biological replicates of quantitative PCR were performed for each gene. The relative transcript level of each gene was normalized to that of ACTIN cDNA for quantitation.
Ren W., Wu F., Bai J., Li X., Yang X., Xue W., Liu H, & He Y. (2020). BcpLH organizes a specific subset of microRNAs to form a leafy head in Chinese cabbage (Brassica rapa ssp. pekinensis). Horticulture Research, 7, 1.
Full text: Click here
Publication 2020
Actins austin Biopharmaceuticals Biotin Buffers Chloroform Crossbreeding Deoxyribonuclease I DNA, Complementary DNA Contamination DNA Probes Electrophoresis Electrophoretic Mobility Shift Assay Ethanol Gels Genes isopentyl alcohol Light MicroRNAs Northern Blot Oligonucleotide Primers Oligonucleotides Phenol Phosphorus Plant Leaves Plants Real-Time Polymerase Chain Reaction RNA, Plant RNA-Directed DNA Polymerase Tissue, Membrane Tissues Tris-borate-EDTA buffer trizol

Most recents protocols related to «Northern Blot»

1
MS2-micF Affinity Purification and Analysis
The MAPS was performed as described before [19 (link),43 ,44 (link)]. The rne131 background was used to maximize target recovery. Cells grown in the LB medium were harvested at OD600nm = 0.5 and 1.0 following induction of either pBAD-micF or pBAD-MS2-micF expression with 0.1% arabinose for 15 min. Raw data was analysed as described previously, with the only modification being that due to the low quality of reads in the R2 sequencing dataset, analysis was done single-ended using R1 [43 ,44 (link)]. Selection of the putative targets was done according to the following cut-offs: RNAs with >25 reads (before normalization) and a ratio ≥3 were selected. Among these, only the RNAs with >50 reads (before normalization) and a ratio of <6.5 in the pBAD-MS2 control were selected (Supplementary Data Table S4). GalaxyProject [45 (link)] and UCSC Microbial Genome Browser [46 (link)] were used to analyse and visualize the data. MS2‐micF GEO accession number is GSE113584. MS2-control was previously published with GEO accession number GSE67606 [47 (link)]. For MS2 affinity purification, followed by Northern blot analysis in different backgrounds (WT, ΔgcvB, hfq Y25D), cells were harvested at OD600nm = 2.0. Input samples were taken prior to any manipulation of the culture. The output samples result from MS2 affinity purification. RNA was analysed by Northern blot as described below.
Carrier M.C., Lalaouna D, & Massé E. (2023). Hfq protein and GcvB small RNA tailoring of oppA target mRNA to levels allowing translation activation by MicF small RNA in Escherichia coli. RNA Biology, 20(1), 59-76.
Full text: Click here
Publication 2023
Arabinose Cells Chromatography, Affinity Genome, Microbial Microtubule-Associated Proteins Northern Blot Northern Blotting
2
Synechocystis 6803 Total DNA Extraction and Northern Blot
Total DNA was prepared from Synechocystis 6803 by collecting cells from 50 ml cultures at an OD750 of 1–1.2 by centrifugation at 3,237 g for 10 min. The pellets were resuspended in 1 ml SET buffer (50 mM Tris, pH 7.5; 1 mM EDTA, 25% (w/v) sucrose). For lysis, resuspended cells were incubated in 100 mM EDTA (pH 8), 2% SDS (w/v) and 100 μg/ml proteinase K at 50°C for 16 h. DNA was extracted by phenol/chloroform/isoamylalcohol (25:24:1) extraction. DNA was precipitated from the final aqueous phase by adding 1 vol isopropanol, incubation at-20°C for at least 2 h and centrifugation at 15,871 g for 30 min at 4°C. The pellet was washed with 70% ethanol, air-dried and resuspended in 30 μl sterile Milli-Q water.
For restriction analysis, 4 μg of total DNA each were digested with FastDigest HindIII restriction endonuclease for 3 h at 37°C to ensure complete digestion. The digested DNA samples were subjected to gelelectrophoretic separation for 1 to 2 h at room temperature on ethidium bromide stained 0.8% agarose gels with 120 V. Thereafter, the gels were incubated at 70 rpm at room temperature for 30 min each in 0.25 M HCl, in denaturation solution (1.5 M NaCl, 0.5 M NaOH) and in neutralization solution (1.5 M NaCl, 0.5 M Tris pH 7.5) prior to blotting. The DNA samples were blotted onto Hybond-N+ nylon membranes (Cytiva) by capillary transfer overnight with 20x SSC (3 M NaCl, 300 mM sodium citrate pH 7) as transfer buffer. After blotting the DNA was crosslinked to the membrane with 120 mJ using a UV-Stratalinker (Stratagene).
For the generation of isotope-labelled probes, templates were amplified via PCR, using the primers P38 and P40 containing the T7 promoter sequence together with the respective primers P37 and P39 and genomic DNA from Synechocystis 6803. Transcript probes labelled with [α-32P]-UTP (3,000 Ci/mmol, 10 mCi/ml) were generated from these templates using the MAXIscript® T7 In Vitro Transcription Kit (Thermo Fisher Scientific).
Hybridization was performed overnight at 52°C in hybridization buffer followed by 10 min wash steps each in wash buffers 1, 2 and 3 (for buffers see Northern blot section) at 47°C. The signals were detected with a Phosphor Imaging Screen (Bio-Rad) and the GE Healthcare Typhoon FLA 9500 imaging system.
Kaltenbrunner A., Reimann V., Hoffmann U.A., Aoyagi T., Sakata M., Nimura-Matsune K., Watanabe S., Steglich C., Wilde A, & Hess W.R. (2023). Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA. Frontiers in Microbiology, 14, 1112307.
Full text: Click here
Publication 2023
Acid Hybridizations, Nucleic Buffers Capillaries Cells Centrifugation Chloroform Deoxyribonuclease HindIII Digestion Edetic Acid Endopeptidase K Ethanol Ethidium Bromide Gels Genome Isopropyl Alcohol Isotopes Northern Blot Nylons Oligonucleotide Primers Pellets, Drug Phenols Phosphorus Sepharose Sodium Chloride Sodium Citrate Sterility, Reproductive Sucrose Synechocystis Tissue, Membrane Transcription, Genetic Tromethamine Typhoons
3
Quantification of Protein and RNA Levels
The levels of protein and RNA were verified by Western and Northern blots, respectively. In brief, cultured cells were harvested with trypsinization and lysed by RIPA lysis buffer (25 mM Tris–HCl pH7.6, 150 mM NaCl, 1% NP-40, 1% Sodium deoxycholate, 0.1% SDS) for total protein extraction. Each Western sample contains 30 ug total protein that was denatured in loading buffer (50 mM Tris·HCl, pH 6.8, 2% SDS, 6% Glycerol, 2 mM DTT, 0.01% Bromophenol Blue) by heated at 95 °C for 5 min, followed by separation with denature protein electrophoresis (SDS-PAGE). Separated protein samples were transferred to PVDF membrane, followed by staining with specific primary (anti-c-myc, anti-beta-actin, anti-Fluc, anti-Rluc, anti-EGFP, etc.) and HRP-conjugated secondary (anti-rabbit or anti-mouse) antibodies. RNA samples were prepared following the protocol described by Mordstein et al. (2020 (link)) with TRIzol™ Reagent (Invitrogen, USA). Cytosolic fractions of RNAs were separated with denature agarose electrophoresis, followed by transferring to nylon blotting membranes. Transferred membrane were probed with digoxigenin (DIG) labelled Fluc- or Rluc-specific probes and then documented by chemiluminescence imager.
Chen S.C., Xu C.T., Chang C.F., Chao T.Y., Lin C.C., Fu P.W, & Yu C.H. (2023). Optimization of 5′UTR to evade SARS-CoV-2 Nonstructural protein 1-directed inhibition of protein synthesis in cells. Applied Microbiology and Biotechnology, 107(7-8), 2451-2468.
Publication 2023
Antibodies beta-Actin Bromphenol Blue Buffers Chemiluminescence Cultured Cells Cytosol Deoxycholic Acid, Monosodium Salt Digoxigenin Electrophoresis Glycerin Mus Nonidet P-40 Northern Blot Nylons polyvinylidene fluoride Proteins Rabbits Radioimmunoprecipitation Assay RNA SDS-PAGE Sepharose Sodium Chloride Tissue, Membrane trizol Tromethamine
4
Quantification of Small RNA Molecules
DIG-based northern blot and stem-loop RT-qPCR were done as described in ref. 33 (link) and 32 (link), respectively. RT-PCR for full-length tRNAs was done using Superscript IV at 50 °C. Probes, primers, and assay conditions are provided in SI Appendix, Supplementary Materials and Methods.
Costa B., Li Calzi M., Castellano M., Blanco V., Cuevasanta E., Litvan I., Ivanov P., Witwer K., Cayota A, & Tosar J.P. (2023). Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids. Proceedings of the National Academy of Sciences of the United States of America, 120(4), e2216330120.
Publication 2023
Biological Assay Northern Blot Oligonucleotide Primers Reverse Transcriptase Polymerase Chain Reaction Stem, Plant Transfer RNA
5
Determining RNA Half-Lives in Biofluids
For determination of RNA half-lives in biofluids, 1 μg heated and refolded U2-OS total RNA was incubated at 37 °C for variable periods with 50 μL undiluted or PBS-diluted biofluid samples. Digested RNAs were purified by SPE and analyzed by northern blot.
Costa B., Li Calzi M., Castellano M., Blanco V., Cuevasanta E., Litvan I., Ivanov P., Witwer K., Cayota A, & Tosar J.P. (2023). Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids. Proceedings of the National Academy of Sciences of the United States of America, 120(4), e2216330120.
Publication 2023
Northern Blot RNA U2 small nuclear RNA

Top products related to «Northern Blot»

1
Trizol reagent by Thermo Fisher Scientific
Sourced in United States, China, Japan, Germany, United Kingdom, Canada, France, Italy, Australia, Spain, Switzerland, Netherlands, Belgium, Lithuania, Denmark, Singapore, New Zealand, India, Brazil, Argentina, Sweden, Norway, Austria, Poland, Finland, Israel, Hong Kong, Cameroon, Sao Tome and Principe, Macao, Taiwan, Province of China, Thailand
TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
2
Trizol by Thermo Fisher Scientific
Sourced in United States, Germany, China, Japan, United Kingdom, Canada, France, Italy, Spain, Australia, Switzerland, Belgium, Denmark, Netherlands, India, Ireland, Lithuania, Singapore, Sweden, Norway, Austria, Brazil, Argentina, Hungary, Sao Tome and Principe, New Zealand, Hong Kong, Cameroon, Philippines
TRIzol is a monophasic solution of phenol and guanidine isothiocyanate that is used for the isolation of total RNA from various biological samples. It is a reagent designed to facilitate the disruption of cells and the subsequent isolation of RNA.
3
Dig northern starter kit by Roche
Sourced in Switzerland, United States, Germany, China
The DIG Northern Starter Kit is a laboratory equipment product designed for the detection and analysis of specific RNA molecules in northern blot experiments. It provides the necessary components to perform this technique, including buffers, reagents, and membranes.
4
Hybond n membrane by GE Healthcare
Sourced in United Kingdom, United States, Sweden, Germany, Japan, Canada
Hybond-N+ membrane is a nylon-based membrane used for nucleic acid transfer and immobilization in molecular biology applications. It provides a stable surface for the binding and detection of DNA, RNA, and other nucleic acid samples. The membrane is designed to offer high binding capacity and efficient capillary transfer of nucleic acids.
5
Northernmax kit by Thermo Fisher Scientific
Sourced in United States, Japan
The NorthernMax Kit is a laboratory equipment product designed for Northern blot analysis. It provides the necessary reagents and materials to perform this well-established RNA detection and quantification technique.
6
Hybond n membrane by Cytiva
Sourced in United Kingdom, United States, Sweden, Germany, China, Canada
Hybond-N+ membrane is a nylon-based membrane used for DNA and RNA transfer and immobilization in molecular biology applications. It provides a stable and efficient platform for blotting and hybridization experiments.
7
Tri reagent by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, Australia, Israel, France, Sao Tome and Principe, Spain, Japan, Canada, Macao, India, Poland, Switzerland, Netherlands, Czechia, China, Ireland, Denmark, Austria, Sweden, New Zealand, Palestine, State of, Estonia, Portugal, Cameroon
TRI Reagent is a single-step liquid extraction reagent used for the isolation of total RNA, DNA, and proteins from a wide range of biological samples. It is a mixture of phenol and guanidine isothiocyanate in a monophasic solution.
8
Hybond n nylon membrane by Cytiva
Sourced in United Kingdom, United States, Germany, Sweden
Hybond-N+ is a positively charged nylon membrane designed for nucleic acid transfer and immobilization. It provides efficient binding and retention of both DNA and RNA samples. The membrane features a smooth surface and uniform pore size for consistent results.
9
Hybond n by GE Healthcare
Sourced in United Kingdom, United States, Germany, Japan
Hybond-N is a nylon-based membrane used in nucleic acid hybridization and transfer applications. It provides a high-binding capacity for both DNA and RNA, allowing for efficient immobilization and detection of these biomolecules.
10
Chemiluminescent nucleic acid detection module by Thermo Fisher Scientific
Sourced in United States, China
The Chemiluminescent Nucleic Acid Detection Module is a laboratory equipment designed to detect and quantify nucleic acids, such as DNA and RNA, based on chemiluminescent signals. The module provides a sensitive and reliable method for analyzing the presence and abundance of specific nucleic acid sequences in various samples.

More about "Northern Blot"

Northern blotting, RNA detection, gene expression analysis, gel electrophoresis, membrane transfer, labeled probes, TRIzol reagent, TRIzol, DIG Northern Starter Kit, Hybond-N+ membrane, NorthernMax Kit, TRI Reagent, Hybond-N+ nylon membrane, Hybond-N, Chemiluminescent Nucleic Acid Detection Module