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Methods in Enzymology 2013To measure the size and amount of RNA transcribed from a specific gene of interest.
To measure the size and amount of RNA transcribed from a specific gene of interest.
Topics: Animals; Blotting, Northern; Electrophoresis, Agar Gel; Nucleic Acid Hybridization; RNA
PubMed: 24034315
DOI: 10.1016/B978-0-12-420037-1.00003-8 -
RNA (New York, N.Y.) Dec 2018Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes....
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
Topics: Blotting, Northern; DNA Probes; Fluorescent Dyes; Nucleic Acid Hybridization; RNA; RNA Probes
PubMed: 30201850
DOI: 10.1261/rna.068213.118 -
Methods in Molecular Biology (Clifton,... 2018microRNAs (miRNAs) have vital roles in regulating gene expression-contributing to major diseases like cancer and heart disease. Over the last decade, thousands of miRNAs...
microRNAs (miRNAs) have vital roles in regulating gene expression-contributing to major diseases like cancer and heart disease. Over the last decade, thousands of miRNAs have been discovered through high throughput sequencing-based annotation. Different classes have been described, as well as a great dynamic range of expression levels. While sequencing approaches provide insight into biogenesis and allow confident identification, there is a need for additional methods for validation and characterization. Northern blotting was one of the first techniques used for studying miRNAs, and remains one of the most valuable as it avoids enzymatic manipulation of miRNA transcripts. Blotting can also provide insight into biogenesis by revealing RNA processing intermediates. Compared to sequencing, however, northern blotting is a relatively insensitive technology. This creates a challenge for detecting low expressed miRNAs, particularly those produced by inefficient, non-canonical pathways. In this chapter, we describe a strategy to study such miRNAs by northern blotting that involves ectopic expression of both miRNAs and miRNA-binding Argonaute (Ago) proteins. Through use of epitope tags, this strategy also provides a convenient method for verification of small RNA competency to be loaded into regulatory complexes.
Topics: Animals; Argonaute Proteins; Blotting, Northern; Gene Expression Regulation; Humans; MicroRNAs; RNA Processing, Post-Transcriptional
PubMed: 29959684
DOI: 10.1007/978-1-4939-8624-8_16 -
International Journal of Molecular... Jun 2019Among a number of mRNA modifications, N6‑methyladenosine (m6A) modification is the most common type in eukaryotes and nuclear‑replicating viruses. m6A has a... (Review)
Review
Among a number of mRNA modifications, N6‑methyladenosine (m6A) modification is the most common type in eukaryotes and nuclear‑replicating viruses. m6A has a significant role in numerous cancer types, including leukemia, brain tumors, liver cancer, breast cancer and lung cancer. Although m6A methyltransferases are essential during RNA modifications, the biological functions of m6A and the underlying mechanisms remain to be fully elucidated, predominantly due to the limited detection methods for m6A. In the present review, the currently available m6A detection methods and the respective scope of their applications are presented to facilitate the further investigation of the roles of m6A in biological process.
Topics: Adenosine; Animals; Biosensing Techniques; Blotting, Northern; Chromatography, High Pressure Liquid; Electrochemical Techniques; Humans; Immunoblotting; Immunoprecipitation; Methylation; Neoplasms; RNA; Sequence Analysis, RNA
PubMed: 31017262
DOI: 10.3892/ijmm.2019.4169 -
Annals of Hematology Feb 2014Testing for the presence of specific cell-surface receptors (such as EGFR or HER2) on tumor cells is an integral part of cancer care in terms of treatment decisions and... (Review)
Review
Testing for the presence of specific cell-surface receptors (such as EGFR or HER2) on tumor cells is an integral part of cancer care in terms of treatment decisions and prognosis. Understanding the strengths and limitations of these tests is important because inaccurate results may occur if procedures designed to prevent false-negative or false-positive outcomes are not employed. This review discusses tests commonly used to identify and characterize cell-surface receptors, such as the erythropoietin receptor (EpoR). First, a summary is provided on the biology of the Epo/EpoR system, describing how EpoR is expressed on erythrocytic progenitors and precursors in the bone marrow where it mediates red blood cell production in response to Epo. Second, studies are described that investigated whether erythropoiesis-stimulating agents could stimulate tumor progression in cancer patients and whether EpoR is expressed and functional on tumor cells or on endothelial cells. The methods used in these studies included immunohistochemistry, Northern blotting, Western blotting, and binding assays. This review summarizes the strengths and limitations of these methods. Critically analyzing data from tests for cell-surface receptors such as EpoR requires understanding the techniques utilized and demonstrating that results are consistent with current knowledge about receptor biology.
Topics: Animals; Blotting, Northern; Blotting, Western; Bone Marrow; Erythroid Cells; Gene Expression Regulation; Humans; Immunohistochemistry; Receptor, ErbB-2; Receptors, Erythropoietin
PubMed: 24337485
DOI: 10.1007/s00277-013-1947-2 -
Viruses Nov 2022Viruses cause severe crop losses. Studying the interaction between viruses and plants is very important for development of control measures. Northern blot is a...
Viruses cause severe crop losses. Studying the interaction between viruses and plants is very important for development of control measures. Northern blot is a well-accepted but very challenging technique to monitor the infection of viruses. Here, we modified the high-molecular-weight (hmw)RNA Northern blot experiment process, utilizing vertical electrophoresis to separate the RNA with denatured agarose gel. This protocol is compatible with regular equipment for Western blots and small RNA Northern blots and requires less input of total RNA. A new method to label the probe with biotin was also developed, which requires commonly used T4 DNA polymerase and detects viral RNA with high sensitivity. These new protocols made hmwRNA Northern blot cost-effective and easy-to-operate, very suitable for studying virus-host interactions.
Topics: RNA; Biotin; RNA, Viral; Biotinylation; Nucleic Acid Hybridization; Blotting, Northern
PubMed: 36560668
DOI: 10.3390/v14122664 -
BMC Molecular Biology Apr 2011Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide additions. The...
BACKGROUND
Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide additions. The scale of miRNA end-heterogeneity is best shown by deep sequencing data revealing not only the major miRNA variants but also those that occur in only minute amounts and are unlikely to be of functional importance. All RNA interference (RNAi) technology reagents that are expressed and processed in cells are also exposed to the same machinery generating end-heterogeneity of the released short interfering RNAs (siRNAs) or miRNA mimetics.
RESULTS
In this study we have analyzed endogenous and exogenous RNAs in the range of 20-70 nt by high-resolution northern blotting. We have validated the results obtained with northern blotting by comparing them with data derived from miRNA deep sequencing; therefore we have demonstrated the usefulness of the northern blotting technique in the investigation of miRNA biogenesis, as well as in the characterization of RNAi technology reagents.
CONCLUSIONS
The conventional northern blotting enhanced to high resolution may be a useful adjunct to other miRNA discovery, detection and characterization methods. It provides quantitative data on distribution of major length variants of abundant endogenous miRNAs, as well as on length heterogeneity of RNAi technology reagents expressed in cells.
Topics: Animals; Blotting, Northern; HEK293 Cells; Humans; Mice; MicroRNAs; RNA Interference; RNA Precursors; RNA, Small Interfering; Sequence Analysis, RNA
PubMed: 21481235
DOI: 10.1186/1471-2199-12-14 -
BMC Genomics Jan 2022Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages...
BACKGROUND
Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use.
RESULTS
In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment.
CONCLUSIONS
The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA.
Topics: Blotting, Northern; Formaldehyde; Nucleic Acid Hybridization; RNA; RNA, Messenger
PubMed: 35057752
DOI: 10.1186/s12864-021-08275-w -
Nature Communications Sep 2023Small RNAs (sRNAs) within 15-30 nt such as miRNA, tsRNA, srRNA with 3'-OH have been identified. However, whether these sRNAs are the major 15-30 nt sRNAs is still...
Small RNAs (sRNAs) within 15-30 nt such as miRNA, tsRNA, srRNA with 3'-OH have been identified. However, whether these sRNAs are the major 15-30 nt sRNAs is still unknown. Here we show about 90% mammalian sRNAs within 15-30 nt end with 2',3'-cyclic phosphate (3'-cP). TANT-seq was developed to simultaneously profile sRNAs with 3'-cP (sRNA-cPs) and sRNA-OHs, and huge amount of sRNA-cPs were detected. Surprisingly, sRNA-cPs and sRNA-OHs usually have distinct sequences. The data from TANT-seq were validated by a novel method termed TE-qPCR, and Northern blot. Furthermore, we found that Angiogenin and RNase 4 contribute to the biogenesis of sRNA-cPs. Moreover, much more sRNA-cPs than sRNA-OHs bind to Ago2, and can regulate gene expression. Particularly, snR-2-cP regulates Bcl2 by targeting to its 3'UTR dependent on Ago2, and subsequently regulates apoptosis. In addition, sRNA-cPs can guide the cleavage of target RNAs in Ago2 complex as miRNAs without the requirement of 3'-cP. Our discovery greatly expands the repertoire of mammalian sRNAs, and provides strategies and powerful tools towards further investigation of sRNA-cPs.
Topics: Animals; MicroRNAs; 3' Untranslated Regions; Apoptosis; Blotting, Northern; Mammals
PubMed: 37723159
DOI: 10.1038/s41467-023-41554-6 -
RNA (New York, N.Y.) Mar 2022The 22 mitochondrial and ∼45 cytosolic tRNAs in human cells contain several dozen different post-transcriptional modified nucleotides such that each carries a unique...
The 22 mitochondrial and ∼45 cytosolic tRNAs in human cells contain several dozen different post-transcriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression, and deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our laboratory developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ∼10 yr ago, which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of a DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This ositive ybridization in the bsence of odification (PHAM) assay has proven useful for iA37, tA37, mC32, and mG26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.
Topics: Blotting, Northern; HEK293 Cells; Humans; Methylation; RNA Processing, Post-Transcriptional; RNA, Transfer; Yeasts
PubMed: 34930808
DOI: 10.1261/rna.078929.121