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How to detect specific mRNA dsRNA by Northern analysis?

How to detect specific mRNA dsRNA by Northern analysis?



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  1. I silenced a gene by RNAi method, for that i chose the gene and the size of the gene is 1.5kb and my insert size is 500nts. i did the transfection and i want to confirm the silencing by northern analysis. when i did i got the ribosomal RNA instead of my specific mRNA reduction and dsRNA accumulation.

  2. Total RNA was prepared with TRIzol reagent (Sigma), and 20 μg/lane was fractionated on a 1.2% agarose, 2.2 M formaldehyde gel. The RNA was visualized with ethidium bromide. The RNA was transferred to a nylon membrane (Hybond; Amersham Biosciences) for 14 hrs and probed with [α-32P] UTP-labeled used SSC for transfer and Prehybridization at 55 for 2hrs incubation, in the probe for 10hrs and Washing at 60 for 2X SSC 20 mins two times.

In this image you can the samples TET induced and uninduced RNA. but the result was confusing and am getting such a strong ribosomal RNA (2.2 Kb) contamination.


Current approaches to micro-RNA analysis and target gene prediction

It is becoming increasingly evident that micro-RNAs (miRNA) play a significant role in regulating the cellular machinery. These ∼22-nt non-coding RNAs function as negative regulators of gene expression. Since their discovery, considerable information has been obtained on miRNA biology and the mechanism of their action. Guidelines have been established for miRNA nomenclature and databases have been built to house all miRNA from many species. A number of methodologies are available for miRNA analysis. There is a lot of interest in developing bioinformatics approaches to predict miRNA target genes. This article will bring together the information on our current knowledge of miRNA biology, the approaches for miRNA analysis, and computational strategies to gain insight in miRNA functional roles.

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Author Summary

RNA interference (RNAi) is a cellular mechanism activated by double-stranded RNA (dsRNA). Cellular dsRNA-specific RNaseIII enzymes (Dicer) recognize dsRNA and process it into double-stranded small interfering RNAs (ds-siRNAs) of 21–25 nucleotides (nt). siRNAs guide RNAi to degrade also single-stranded RNA homologous to the trigger. RNAi regulates gene expression, controls transposons, and represents an important antiviral defense mechanism. Therefore, viruses encode proteins dedicated to countering RNAi. In this study, the RNaseIII enzymes of a fish DNA virus (PPIV) and a plant RNA virus (SPCSV) were compared for suppression of RNAi in non-host organisms. The fish iridovirus RNaseIII suppressed RNAi in a plant and a nematode. It also enhanced accumulation of an RNAi suppressor deficient virus in plants, and suppressed antiviral RNAi and could rescue multiplication of an unrelated, RNAi suppressor-defective virus in nematodes. In contrast, the plant virus RNaseIII could suppress RNAi only in plants. Our results underscore that the active viral RNaseIII enzymes suppress RNAi. Their activity in suppression of RNAi seems to differ for the spectrum of unrelated organisms. Understanding of this novel mechanism of RNAi suppression may inform means of controlling the diseases and economic losses which the RNaseIII-containing viruses cause in animal and plant production.

Citation: Weinheimer I, Jiu Y, Rajamäki M-L, Matilainen O, Kallijärvi J, Cuellar WJ, et al. (2015) Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants. PLoS Pathog 11(3): e1004711. https://doi.org/10.1371/journal.ppat.1004711

Editor: Hui-Shan Guo, Institute of Microbiology of the Chinese Academy of Sciences, CHINA

Received: August 31, 2014 Accepted: January 28, 2015 Published: March 6, 2015

Copyright: © 2015 Weinheimer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was funded by the Academy of Finland (1134335, 1253126 and 1276136 to JPTV, 133552 to JJ, and 259797 to CIH), Lundbeck Foundation (to MS) and The National Institute of Health (1R03AI092159-01A1 to RL).IW was supported by the Viikki Doctoral Program in Molecular Biosciences (VGSB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


Materials and methods

Rr-Luc mRNA consisted of the 926-nucleotide Rrluciferase coding sequence flanked by 25 nucleotides of 5′ untranslated sequence from the pSP64 plasmid polylinker and 25 nucleotides of 3′ untranslated sequence consisting of 19 nucleotides of pSP64 plasmid polylinker sequence followed by a 6-ntSacI site. PP-Luc mRNA contained the 1653-nt Pp luciferase coding sequence with a KpnI site introduced immediately before the Pp luciferase stop codon. ThePp coding sequence was flanked by 5′ untranslated sequences consisting of 21 nt of pSP64 plasmid polylinker followed by 512 nt of the 5′ untranslated region (UTR) from the Drosophila hunchback mRNA and 3′ untranslated sequences consisting of the 562-nt hunchback 3′ UTR followed by a 6-nt SacI site. The hunchback 3′ UTR sequences used contained six G-to-U mutations that disrupt function of the Nanos Response Elements in vivo and in vitro (D. Chagnovich, P.D. Zamore, R. Lehman, and D.P. Bartel, unpubl.). Both reporter mRNAs terminated in a 25-nt poly(A) tail encoded in the transcribed plasmid. For bothRr-Luc and Pp-Luc mRNAs, the transcripts were generated by run-off transcription from plasmid templates cleaved at anNsiI site that immediately followed the 25-nt-encoded poly(A) tail. To ensure that the transcripts ended with a poly(A) tail, theNsiI-cleaved transcription templates were resected with T4 DNA Polymerase in the presence of dNTPs. The SP6 mMessage mMachine kit (Ambion) was used for in vitro transcription. With this kit, ∼80% of the resulting transcripts are 7-methyl guanosine capped. 32 P-radiolabeling was accomplished by including [α- 32 P]UTP in the transcription reaction.

For Pp-Luc, ssRNA, asRNA, and dsRNA corresponded to positions 93–597 relative to the start of translation, yielding a 505-bp dsRNA. For Rr-Luc, asRNA, ssRNA, and dsRNA corresponded to positions 118–618 relative to the start of translation, yielding a 501-bp dsRNA. The Drosophila nanos competitor dsRNA corresponded to positions 122–629 relative to the start of translation, yielding a 508-bp dsRNA. ssRNA, asRNA, and dsRNA (diagrammed in Fig. A) were transcribed in vitro with T7 RNA polymerase from templates generated by the PCR. After gel purification of the T7 RNA transcripts, residual DNA template was removed by treatment with RQ1 DNase (Promega). The RNA was extracted with phenol and chloroform, and then precipitated and dissolved in water.

RNA annealing and native gel electrophoresis

ssRNA and asRNA (0.5 μ m ) in 10 m m Tris-HCl (pH 7.5) with 20 m m NaCl were heated to 95°C for 1 min, then cooled and annealed at room temperature for 12–16 hr. The RNAs were precipitated and resuspended in lysis buffer (below). To monitor annealing, RNAs were electrophoresed in a 2% agarose gel in TBE buffer and stained with ethidium bromide (Sambrook et al. 1989).

Lysate preparation

Zero- to 2-hr-old embryos from Oregon R flies were collected on yeasted molasses agar at 25°C. Embryos were dechorionated for 4–5 min in 50% (vol/vol) bleach, washed with water, blotted dry, and transferred to a chilled Potter-Elvehjem tissue grinder (Kontes). Embryos were lysed at 4°C in 1 ml of lysis buffer (100 m m potassium acetate, 30 m m HEPES-KOH at pH 7.4, 2 m m magnesium acetate) containing 5 m m dithiothreitol (DTT) and 1 mg/ml Pefabloc SC (Boehringer Mannheim) per gram of damp embryos. The lysate was centrifuged for 25 min at 14,500g at 4°C, and the supernatant flash frozen in aliquots in liquid nitrogen and stored at −80°C.

Reaction conditions

Lysate preparation and reaction conditions were derived from those described by Hussain and Leibowitz (1986). Reactions contained 50% (vol/vol) lysate, mRNAs (10–50 p m final concentration), and 10% (vol/vol) lysis buffer containing the ssRNA, asRNA, or dsRNA (10 n m final concentration). Each reaction also contained 10 m m creatine phosphate, 10 μg/ml creatine phosphokinase, 100 μ m GTP, 100 μ m UTP, 100 μ m CTP, 500 μ m ATP, 5 m m DTT, 0.1 U/μL RNasin (Promega), and 100 μ m of each amino acid. The final concentration of potassium acetate was adjusted to 100 m m . For standard conditions, the reactions were assembled on ice and then preincubated at 25°C for 10 min before adding mRNA. After adding mRNAs, the incubation was continued for an additional 60 min. The 10-min preincubation step was omitted for the experiments in Figures and . Reactions were quenched with 4 volumes of 1.25× Passive Lysis Buffer (Promega). Pp andRr luciferase activity was detected in a Monolight 2010 Luminometer (Analytical Luminescence Laboratory) with the Dual-Luciferase Reporter Assay System (Promega).

RNA stability

Reactions with 32 P-radiolabeled mRNA were quenched by the addition of 40 volumes of 2× PK buffer (200 m m Tris-HCl at pH 7.5, 25 m m EDTA, 300 m m NaCl, 2% wt/vol sodium dodecyl sulfate). Proteinase K (E.M. Merck dissolved in water) was added to a final concentration of 465 μg/ml. The reactions were then incubated for 15 min at 65°C, extracted with phenol/chloroform/isoamyl alcohol (25:24:1), and precipitated with an equal volume of isopropanol. Reactions were analyzed by electrophoresis in a formaldehyde/agarose (0.8% wt/vol) gel (Sambrook et al. 1989). Radioactivity was detected by exposing the agarose gel [dried under vacuum onto Nytran Plus membrane (Amersham)] to an image plate (Fujix) and quantified with a Fujix Bas 2000 and Image Gauge 3.0 (Fujix) software.

Commercial lysates

Untreated rabbit reticulocyte lysate (Ambion) and wheat germ extract (Ambion) reactions were assembled according to the manufacturer's directions. dsRNA was incubated in the lysate at 27°C (wheat germ) or 30°C (reticulocyte lysate) for 10 min prior to the addition of mRNAs.


RESULTS AND DISCUSSION

LNAs comprise a new class of bicyclic high-affinity RNA analogues in which the furanose ring in the sugar–phosphate backbone is chemically locked in an N-type (C3′- endo ) conformation by the introduction of a 2′- O ,4′- C methylene bridge ( 15 , 16 ). Several studies have demonstrated that LNA-modified oligonucleotides exhibit unprecedented thermal stability when hybridized with their DNA and RNA target molecules ( 16 – 22 ). Consequently, an increase in melting temperature ( Tm ) of +1–8°C per introduced LNA monomer against complementary DNA, and of +2–10°C per monomer against complementary RNA compared to unmodified duplexes have been reported. Structural studies of different LNA–RNA and LNA–DNA heteroduplexes based on NMR spectroscopy and X-ray crystallography have shown that LNA is an RNA mimic, which fits seamlessly into an A-type Watson–Crick duplex geometry ( 23 – 26 ) similar to that of dsRNA duplexes. Furthermore, in heteroduplexes between LNA oligonucleotides and their complementary DNA oligonucleotides, an overall shift from a B-type duplex towards an A-type duplex has been reported resulting in increased stability of the heteroduplexes. Another important observation is that LNA monomers are also able to twist the sugar conformation of flanking DNA nucleotides from an S-type (C2′- endo ) towards an N-type sugar pucker in LNA-modified DNA oligonucleotides ( 25 – 26 ). The unprecedented thermal stability of LNA oligonucleotides together with their improved mismatch discrimination has made them well suited for highly accurate genotyping assays ( 20 , 27 – 29 ). In addition, LNA-substituted oligonucleotides have been used to increase the sensitivity and specificity in gene expression profiling by spotted oligonucleotide microarrays ( 30 ), in potent and selective gene knock-down by LNA antisense ( 18 – 19 , 31 – 33 ) and more recently, in efficient isolation of intact poly(A) + RNA from lysed cell and tissue extracts by LNA oligo(T) affinity capture ( 21 ).

To exploit the improved hybridization properties of LNAs against complementary RNA targets and the ability of LNA monomers to perturb neighbouring DNA nucleotides in chimeric LNA–DNA oligonucleotides toward the A-type geometry, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants with regular spacing in the LNA substitution pattern ( Table 1 ). First, we synthesized LNA-modified oligonucleotides complementary to the relatively low-abundant mature A.thaliana miR171, by substituting every second and every third nucleotide position with an LNA monomer, designated here as miR171LNA2 and miR171LNA3, respectively ( Table 1 ). Substitution of the miR171 DNA probe with regularly spaced LNAs resulted in significantly increased sensitivity in detecting the mature miR171 in A.thaliana flowers and leaves by 5′-labelled probes, when low stringency washing conditions were employed ( Figure 1 ) in the northern blot analysis, in accordance with their increased thermal duplex stability ( Table 1 ). The larger RNAs detected by both LNA-modified miR171 probes as well as with the DNA probe did not correspond to the expected size of the 123 nt miRNA 171 stem–loop precursor, but most likely represented unspecific hybridization to other A.thaliana RNAs ( Figure 1 ). Although the use of the radioactively labeled miR171LNA2 probe showed increased sensitivity compared to the DNA probe, it also resulted in a high background under the given conditions, presumably due to the high LNA substitution degree (50% LNA) of the probe, which, in turn, resulted in extremely high duplex stability of the LNA–RNA duplexes. In contrast, the miR171LNA3 probe showed significantly improved sensitivity, while simultaneously having a low background on the northern blot comparable to that of the DNA control probe. Consequently, a specific hybridization signal corresponding to the mature A.thaliana miR171 could be obtained after 3 h of exposure with the miR171LNA3 oligonucleotide probe, whereas the DNA control probe required 48 h of exposure in order to obtain a comparable hybridization signal ( Figure 1 ). As expected, the prolonged exposure of the miR171LNA2 and miR171LNA3-probed northern blots resulted in highly over-exposed autoradiograms. Similar results were obtained by comparing LNA2- and LNA3-modified probes in the detection of mature miR159 of A.thaliana , except that strong miRNA159-specific signals were obtained with the labeled miR159LNA3 probe already after 1 h of exposure (data not shown). Our findings with the LNA-modified microRNA probes are supported by the NMR spectroscopic studies of different LNA–RNA hybrid structures ( 25 , 26 ). Substitution of a complementary 9mer DNA strand with three LNAs in the resulting nonamer LNA3–RNA hybrid inferred that the number of LNA modifications had reached a saturation level with regard to the structural change into an A-like duplex geometry. Hence, the structural alteration introduced by a fully modified LNA nonamer in the LNA–RNA duplex was found to be small compared to the LNA3–RNA duplex, while the Tm of this duplex was significantly increased ( 26 ). Similarly, the calculated Tm values of our A.thaliana LNA2-modified miRNA probes were higher compared to the LNA3 probes, requiring further optimization of the hybridization and washing conditions in northern blot analysis, as demonstrated by the high background on the northerns. In contrast, the LNA3-modified miRNA probes could be readily used in standard northern blots without further adjustment of the hybridization conditions. We thus decided to use LNA3-substituted microRNA probes in all subsequent comparative experiments.

Comparison of LNA2- and LNA3-modified oligonucleotide probes with DNA probes in the detection of the low-abundant miR171 in A.thaliana flowers and leaves by northern blot analysis. Total RNAs (40 μg/sample) from A.thaliana flowers (lane 1) or leaves (lane 2) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR171LNA2 ( A ), miR171LNA3 ( B ) and miR171DNA ( C ) oligonucleotide probes at 37°C. The membranes were washed at low stringency. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels). M denotes the RNA molecular weight marker of 20–80 nucleotides.

Comparison of LNA2- and LNA3-modified oligonucleotide probes with DNA probes in the detection of the low-abundant miR171 in A.thaliana flowers and leaves by northern blot analysis. Total RNAs (40 μg/sample) from A.thaliana flowers (lane 1) or leaves (lane 2) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR171LNA2 ( A ), miR171LNA3 ( B ) and miR171DNA ( C ) oligonucleotide probes at 37°C. The membranes were washed at low stringency. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels). M denotes the RNA molecular weight marker of 20–80 nucleotides.

The sensitivity in detection of mature microRNAs by northern blots by LNA3-modified oligonucleotide probes was assessed by loading serial dilutions of the same A.thaliana total RNA sample from 100 to 2.5 μg on the northern gels, followed by hybridization with 5′ end-labelled miR171LNA3, miR171DNA, miR319LNA3 and miR319DNA probes, respectively ( Figure 2 ). The use of LNA3 probes resulted in significantly increased sensitivity for mature A.thaliana miR171 and miR319, in accordance with our initial results, showing a readily detectable signal in 2.5 μg of total RNA. The average increase in sensitivity was estimated to be at least 10-fold compared to the corresponding DNA control probes ( Figure 2 ).

Assessment of the sensitivity of LNA3-modified probes compared with DNA probes in the detection of miR171 and miR319 in A.thaliana flowers by northern blot analysis. Two duplicate dilution series of A.thaliana total RNA from 100 to 2.5 μg were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled DNA ( A ) and LNA3 ( B ) oligonucleotide probes, respectively, at 34°C. The filters were first hybridized with LNA3 and DNA probes specific for mature mir171, washed with low stringency and exposed as indicated. The filters were stripped, exposed for checking that removal of the probes was complete, and then re-hybridized with LNA3 and DNA probes specific for mature miR319, washed with low stringency and exposed as indicated. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

Assessment of the sensitivity of LNA3-modified probes compared with DNA probes in the detection of miR171 and miR319 in A.thaliana flowers by northern blot analysis. Two duplicate dilution series of A.thaliana total RNA from 100 to 2.5 μg were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled DNA ( A ) and LNA3 ( B ) oligonucleotide probes, respectively, at 34°C. The filters were first hybridized with LNA3 and DNA probes specific for mature mir171, washed with low stringency and exposed as indicated. The filters were stripped, exposed for checking that removal of the probes was complete, and then re-hybridized with LNA3 and DNA probes specific for mature miR319, washed with low stringency and exposed as indicated. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

Next, we examined the possibility to use high-stringency hybridization and washing conditions that would allow improved specificity in microRNA detection by northern blots, especially when detecting highly homologous miRNAs, e.g. members of the let-7 microRNA family ( 34 ). Hybridization of the A.thaliana small RNA northern blots with the miR171LNA3 probe resulted in highly specific signals corresponding to the mature miR171 even when using high stringency washing conditions (0.1× SSC, 0.1% SDS at 65°C Figure 3A ). By contrast, the miR171LNA3/2MM probe with two consecutive mismatches in the central positions of the probe ( Table 1 ) and the DNA control oligonucleotide only faintly detected the miR171 on the northern blot ( Figure 3B and C ). Furthermore, the TCVLNA3 control probe detected specifically the virus-derived siRNAs in Turnip crinkle virus (TCV)-infected A.thaliana plants, but no small RNAs in the uninfected plant RNA samples ( Figure 3D ). Taken together, these results indicate that modification of DNA oligonucleotide probes with LNA employing the LNA3 substitution pattern enables the use of high stringency hybridization conditions in the detection of mature microRNAs by northern analysis.

Improved sensitivity and specificity in the detection of miR171 in A.thaliana flowers and leaves by northern blot analysis using an LNA3-modified oligonucleotide probe. Total RNAs (20 μg/sample) from A.thaliana seedlings (lane 1), leaves (lane 2), flowers (lane 3) and TCV (Turnip crinkle virus)-infected leaves (lane 4) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR171LNA3 ( A ), miR171LNA3/2MM ( B ), miR171DNA ( C ) and TCVLNA3 oligonucleotide probes at 42°C ( D ). Stringent washes were carried out in 0.1× SSC, 0.1% SDS at 65°C twice for 5 min. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

Improved sensitivity and specificity in the detection of miR171 in A.thaliana flowers and leaves by northern blot analysis using an LNA3-modified oligonucleotide probe. Total RNAs (20 μg/sample) from A.thaliana seedlings (lane 1), leaves (lane 2), flowers (lane 3) and TCV (Turnip crinkle virus)-infected leaves (lane 4) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR171LNA3 ( A ), miR171LNA3/2MM ( B ), miR171DNA ( C ) and TCVLNA3 oligonucleotide probes at 42°C ( D ). Stringent washes were carried out in 0.1× SSC, 0.1% SDS at 65°C twice for 5 min. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

To further investigate the discriminatory power of LNA3-modified microRNA probes, we synthesized three additional mismatch probes for A.thaliana miR171, miR171LNA3/MM8, miR171LNA3/MM11, miR171LNA3/MM14, having a single mismatch at three different positions of the oligonucleotide sequence ( Table 1 ). Hybridization of the A.thaliana small RNA northern blot resulted in significantly decreased signals for the mature miRNA171 by all three mismatch probes compared to the perfect match probe although at somewhat different extent, whereas no signals were detected by the double mismatch probe ( Figure 4 ). By using high stringency washing conditions, the hybridization signal obtained by the single mismatch probes could be decreased even further, whereas the signal obtained with the perfect match probe miR171LNA3 remained unchanged under the same conditions ( Figure 4 ). This is in good agreement with previous reports for improved mismatch discrimination by LNA probes ( 20 , 27 – 29 ), and implies that the microRNA detection by labelled, LNA3-substituted probes is highly specific.

Assessment of the specificity of LNA3-modified probes using perfect match and different mismatched probes in the detection of miR171 in A.thaliana flowers and leaves by northern blot analysis. Total RNAs (20 μg/sample) from A.thaliana flowers (lane 1) and leaves (lane 2) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR171LNA3 ( A ), miR171LNA3/2MM ( B ), miR171LNA3/MM11 ( C ), miR171LNA3/MM8 ( D ) and miR171LNA3/MM14 ( E ) LNA-modified oligonucleotide probes at 45°C. The filters were washed at low stringency (upper panels) and high stringency (middle panels). The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels). ( F ) Validation of the perfect match and the different mismatched LNA probes using complementary DNA oligonucleotide targets. The end-labelled LNA-modified probes were hybridized to their respective perfect match DNA oligonucleotide targets as well as a DNA oligonucleotide corresponding to the mature miRNA171 sequence.

Assessment of the specificity of LNA3-modified probes using perfect match and different mismatched probes in the detection of miR171 in A.thaliana flowers and leaves by northern blot analysis. Total RNAs (20 μg/sample) from A.thaliana flowers (lane 1) and leaves (lane 2) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR171LNA3 ( A ), miR171LNA3/2MM ( B ), miR171LNA3/MM11 ( C ), miR171LNA3/MM8 ( D ) and miR171LNA3/MM14 ( E ) LNA-modified oligonucleotide probes at 45°C. The filters were washed at low stringency (upper panels) and high stringency (middle panels). The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels). ( F ) Validation of the perfect match and the different mismatched LNA probes using complementary DNA oligonucleotide targets. The end-labelled LNA-modified probes were hybridized to their respective perfect match DNA oligonucleotide targets as well as a DNA oligonucleotide corresponding to the mature miRNA171 sequence.

Finally, we demonstrated the utility of the LNA3-modified miRNA oligonucleotide probes in characterizing expression patterns of five different microRNAs, the mouse miR122a, miR124 and miR128, and plant miR161 and miR167, respectively. Total RNA samples extracted from mouse brain and liver, and A.thaliana and N.benthamiana flowers and leaves were subjected to northern blot analyses and hybridized with the different LNA3-modified probes. Highly specific signals corresponding to the mature mouse miR124 and miR128 were detected in the mouse brain, but not in the liver ( Figure 5B and C ), while miR122a was detected only in the liver RNA sample ( Figure 5A ), which is in good agreement with previous reports ( 35 ). No unspecific background signals were detected using TCVLNA3 probe as a negative control ( Figure 5D ). The mature miR167 was detected both in A.thaliana and N.benthamiana flowers and leaves ( Figure 5E ), whereas our results revealed that the presence of miR161 was specific for A.thaliana tissues since no signal was detected in samples originating from N.benthamiana ( Figure 5F ).

Northern blot analysis of three mouse and two A.thaliana microRNAs using LNA3-modified oligonucleotide probes. (A–D) Total RNAs (20 μg/sample) from mouse brain (lane 1), liver (lane 2) and A.thaliana flowers (lane 3) were electrophoresed on 12% polyacrylamide gels under denaturing conditions, blotted and hybridized with 32 P-labelled miR122aLNA3 ( A ), miR128LNA3 ( B ), miR124LNA3 ( C ) LNA probes as well as TCVLNA3 as a negative control probe ( D ) at 45°C. ( E and F ) Total RNAs (20 μg/sample) from A.thaliana flowers (lane 1), leaves (lane 2) and N.benthamiana flowers (lane 3), leaves (lane 4) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR167LNA3 (E), miR161LNA3 (F) oligonucleotide probes at 45°C. The filters were washed at low stringency. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

Northern blot analysis of three mouse and two A.thaliana microRNAs using LNA3-modified oligonucleotide probes. (A–D) Total RNAs (20 μg/sample) from mouse brain (lane 1), liver (lane 2) and A.thaliana flowers (lane 3) were electrophoresed on 12% polyacrylamide gels under denaturing conditions, blotted and hybridized with 32 P-labelled miR122aLNA3 ( A ), miR128LNA3 ( B ), miR124LNA3 ( C ) LNA probes as well as TCVLNA3 as a negative control probe ( D ) at 45°C. ( E and F ) Total RNAs (20 μg/sample) from A.thaliana flowers (lane 1), leaves (lane 2) and N.benthamiana flowers (lane 3), leaves (lane 4) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with 32 P-labelled miR167LNA3 (E), miR161LNA3 (F) oligonucleotide probes at 45°C. The filters were washed at low stringency. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

In conclusion, we report here that partial substitution of DNA oligonucleotides by LNA results in significantly improved sensitivity in detecting mature microRNAs by northern blot analysis, while simultaneously being highly specific. Because the electrostatic properties of LNA probes are similar to DNA and RNA oligonucleotides, and the fact that LNA chemistry is fully compatible with DNA phosphoramidite chemistry, makes it easy to synthesize LNA oligonucleotide probes using standard phosphoramidite chemistry and oligonucleotide purification methods. Another important practical advantage is that the LNA-modified oligonucleotides are fully soluble in water, which makes their labelling and use in nucleic acid hybridization experiments simple ( 16 , 17 ). By modifying DNA oligonucleotides with LNAs using the LNA3 substitution pattern, in which every third nucleotide position is modified by the corresponding LNA nucleotide, we were able to use the probes in northern blot analysis employing standard end-labelling methods and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific as demonstrated by the use of mismatch LNA3 probes. Besides being highly efficient as northern probes, the same LNA3-modified oligonucleotide probes could also be useful for addressing the spatial expression of microRNAs by in situ hybridization as well as expression profiling by spotted microarrays using different LNA-modified capture probes designed to detect mature and precursor microRNAs.

We wish to thank Marianne Bonde Mogensen and Mette Bjørn at Exiqon for excellent technical assistance. This research was supported by a grant from the Hungarian Scientific Research Fund (OTKA T038313) and a grant from the European Commission as part of the RIBOREG EU FP6 project (LSHG-CT-2003503022).


Experimental procedures

Construction of binary RNAi hairpin vector and rice transformation

Interfering sequences for MsChi1 (accession No. AY508698.1) and MsChi2 (accession No. AB201283.1) were amplified from M. separata gut tissue cDNA and inserted as inverted repeats into the pCB2004B binary vector by gateway cloning to generate a hairpin RNAi vector. All the primers used in this study are listed in Table S3. Recombinant vectors were introduced into Agrobacterium tumefaciens strain EHA105, and rice (Oryza sativa L. spp. japonica) transformation and selection were performed according to Wuriyanghan et al., 2009 . Positive transformants were screened by Basta selection (20mg/mL BlpR), and transgenic seedlings were confirmed by genomic DNA PCR analysis on the target MsChi1 or MsChi2 gene and the 35S promoter. For Southern blots, five µg of the genomic DNA was digested with the restriction enzyme EcoRI and separated by 0.8% (w/v) agarose gel electrophoresis. The DNA was denatured and transferred to nylon membranes in 20 X SSC solution by capillary transfer and fixed with UV cross-linker. Digoxin (DIG) labelled probes for MsChi1 and MsChi2 were used for hybridization, and the hybridization signal was detected by chemiluminescence method according to Ganbaatar et al., 2017 . The fresh leaves of transgenic rice and control wild-type rice were collected and used for feeding assays.

Insect feeding and bioassays

Laboratory-adapted M. separata eggs were provided by The Institute of Plant Protection (IPP), Chinese Academy of Agricultural Sciences (CAAS). Synchronous third-instar larvae were used in feeding bioassays. The number of surviving larvae was recorded at every other day. After seven days’ feeding, surviving larvae were collected and the body weight and body length were measured. For RT-qPCR experiments on target gene expression and siRNA sequencing, larvae were collected after three days’ feeding and total RNAs were extracted from dissected gut tissues. RNA isolation, RT-PCR, RT-qPCR and northern blot for siRNA detection were performed as Ganbaatar et al., 2017 . Ratio of mRNA abundance between M. separata gene silenced and control sample was calculated by 2 -ΔΔCT method for RT-qPCR. Three biological replicates were performed, and statistical analysis was performed between treated and the control group.

DsRNA/siRNA synthesis, labelling, larvae feeding and bioassays

Interfering sequences for MsActin (accession No. GQ856238.1) were amplified using gene-specific primers containing the T7 promoter sequence 5'-TAATACGACTCACTATAGGGAGA-3' at the 5' end. Three fragments of 241 bp (100–340), 231 bp (1060–1290) and 475 bp (893–1367) were used as the templates to synthesize dsRNAs, namely ds100, ds1060 and ds893, by the 5 X MEGAscript® T7 kit (Cao et al., 2017 ). SiRNA was produced by treatment of ds893 with ShortCut® RNaseIII and was named si893. 100 ng/μl dsRNA or 50 ng/μl siRNA was mixed with artificial diet for feeding experiments. ds893 was chosen for labelling of dsRNA and siRNA. In dsRNA synthesis reactions, UTP was partly replaced with DIG-UTP or Cy3-UTP to obtain labelled dsRNA. Cy3-labelled dsRNA was digested with ShortCut® RNaseIII to obtain Cy3-labelled siRNA. The labelled dsRNA/siRNAs were fed to the larvae. After feeding of DIG-labelled dsRNA for 4 h and 24 h, gut RNA was extracted, resolved on a 1% agarose gel, transferred to nylon membranes and DIG RNA signals on the membrane were visualized with BCIP/NBT alkaline phosphatase substrate solution. After feeding of Cy3-labelled dsRNA or siRNA for 4 h, gut tissue was sectioned, and Cy3 signals and the nuclei were visualized using a 550 nm (Cy3) and 360 nm (DAPI) for excitation and 570 nm (Cy3) and 460 nm (DAPI) for emission under a laser scanning confocal microscope (Carl Zeiss, LSM 710).

Small RNA sequencing, bioinformatic analysis and detection with northern blot hybridization

Three μg of total RNA for each sample was used for generation of small RNA libraries. Synthetic oligonucleotide adapters were ligated to the RNA using T4 RNA ligase. Adapter ligated RNA was reverse transcribed and PCR amplified. PCR products were resolved on 8% polyacrylamide gels, and DNA fragments of 140-160bp were recovered from the gel. The library was sequenced on the Illumina Hiseq 2500 platform and 50bp single-end reads were generated. Small RNAs were mapped to reference sequences by Bowtie2 (version 2.4.1) without mismatches to analyse the target-specific sRNAs. The sorted.bam files resulted from Bowtie2 and samtools (version 1.10) were used for analysis of small RNA size distribution. The small RNAs were visualized by the artemis software (version 18.1.0). Target-specific siRNAs were manually checked via local BLAST, and the numbers of siRNAs were comparatively analysed between treatment and control groups. Small RNA northern blot detection was performed as in the literature using DIG-labelled RNA probes (Ganbaatar et al., 2017 ).

Screening of RNAi machinery genes, evolutionary analysis, RT-PCR and northern blot analysis

M. separata unigenes were obtained by transcriptome sequencing in our previous publication (Liu et al., 2016 ). M. separata RNAi machinery genes were identified from transcriptome data by using homologous gene sequences from other insect species as a query. The protein sequences were multiple compared using ClustalW2. MEGA-X was used to build the phylogenetic tree via the maximum likelihood method, and the bootstrap method was used to verify the quality of evolutionary tree with 1000 times of inspection. Regular PCR was performed using M. separata gut cDNA to detect the presence of the mRNAs for RNAi machinery genes. Northern hybridization experiments were carried out using M. separata gut RNA and 32 P-UTP-labelled negative strand probes according to our previous publication (Diao et al., 2019 ).

Synthesis of PTA expression cassette and transient knockdown analysis in N. benthamiana

We modified PTG (polycistronic-tRNA and CRISPR guide RNA) (Xie et al., 2015 ) to obtain PTA (polycistronic-tRNA-amiR) for silencing of multiple target genes. Gene-specific amiR was designed and synthesized according to literature (Diao et al., 2019 ) using Arabidopsis thaliana miR159b as the backbone. Multiple tRNAs and amiRs sequences were assembled by GoldenGate assembly, amplified with border-specific primers and cloned into the pMD19T vector. KpnΙ/XbaΙ digested PTA fragments were subcloned into the pBI121 vector to produce recombinant PTA vectors. For transient silencing assays, two amiRs for the GUS (β-glucuronidase) and GFP (Green fluorescent protein) genes were designed and assembled into one PTA construct. Recombinant vectors were transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into N. benthamiana leaves together with GFP expressing TMV-GFP or GUS expressing pBI121-GUS vectors. As a control, single amiR constructs for GFP or GUS were used in parallel experiments. Three days post infiltration, target GFP or GUS mRNA expression was evaluated by RT-qPCR using NbActin (accession No. NM_102866.3) as an internal control. GFP fluorescence was also detected using a hand held UV lamp (365 nm).

PTA construct for M. separata gene silencing, maize transformation and insect bioassays

For multiple gene silencing, we chose MsChi2, MsActin and MsREase (Unigene No., Cluster-3827.32653) as the target genes, and the PTA vector was constructed as described above and transformed into Agrobacterium tumefaciens strain EHA101. Maize transformation was performed on immature embryos from the inbred line maize cultivar B104 according to the literature (Vi et al., 2019 Xiang et al., 2018 ). The complete plantlets with healthy roots were transplanted into soil pots and the transgenic seedlings were confirmed using regular PCR with genomic DNA as template. The fresh leaves of transgenic maize and control wild-type maize were collected and used for feeding assays.

Protein expression, purification and biochemical analysis

Protein expression and purification procedures followed the published methods (Wuriyanghan et al., 2009 ). Briefly, the PAZ domain of MsAGO2, the RNaseIII domain of MsDcr2, N-terminal domain of MsSID1 and the whole ORF of MsChi2 were expressed as GST fusions in E. coli strain BL21 (DE3) by pGEX-4T-1 recombinant vectors via IPTG induction. Recombinant GST-tagged proteins were purified and confirmed by SDS-PAGE or protein gel blotting using a mouse anti-GST monoclonal antibody.

Biochemical analysis was performed according to Rivas et al., 2005 . For RNA labelling, ds100 was treated with CIP to dephosphorylate the 5’ ends, purified by phenol/chloroform extraction and end-labelled by 32 P ATP and T4 polynucleotide kinase to obtain 32 P-labelled dsRNA. For ssRNA, 26nt short RNA 5′-GGGCAUCGCCGACCGUAUGCAGAAGG-3′ was end labelled the same way. Non-labelled RNA was used as competitor. Electrophoretic mobility shift assay (EMSA) was performed to investigate the RNA binding ability of MsAGO2 and MsSID1 proteins by using 32 P labelled ssRNA and dsRNA, respectively. The protein/RNA samples were resolved on native TBE polyacrylamide gels and visualized by autoradiography. dsRNA cleavage assays followed the procedures of Li et al, 2015 and Hodgson et al., 2019 . Briefly, 32 P labelled ds100 was mixed with purified MsDcr2, incubated at 37°C for 30 min, and cleavage products were analysed by 15% (v/v) urea PAGE and visualized by phosphorimaging. For chitinase activity assays, 3 mg/ml insect chitin was incubated with purified MsChi2 protein in 20 mM sodium phosphate buffer (pH = 6.0) and incubated at 30°C for different times. The product N-acetylglucosamine was reacted with P-Dimethylaminobenzaldehyde in boiling water for 7 min and centrifuged at 5000 rpm for 10 min, and the supernatant was detected in a microplate at 585 nm (Liu et al., 2017 ).

Accession numbers

MsChi1, AY508698.1 MsChi2, AB201283.1 MsActin, GQ856238.1 NbActin and NM_102866.3. Other M. separata sequences were obtained from transcriptome data (Liu et al., 2016 ).


Measuring gene expression changes on biomaterial surfaces

In situ hybridization and inflammation

Extraction of mRNA and RT-qPCR could be used to get a screen of the types of genes expressed, but this information by itself will be of limited value, as it will not provide the abundance of each cell type. Neutrophils and macrophages are the main cell types that are present in the inflammatory phase of wound healing ( Walker et al., 2016 ) ( Fig. 6.6 ). The balance of neutrophils to macrophages, and further, presence of specific macrophage subpopulations is more pertinent information. The best method for assessing these genes would be in situ hybridization, which uses a labeled complementary RNA or DNA probe to localize a specific DNA or RNA sequence in a section of tissue ( Zhou et al., 2010 ). Using this technique would demonstrate the influence of a biomaterial on the relative number and location of neutrophils and macrophages in the tissue at each specified time point. The advantage of this technique is that it localizes the signal to the specific cells expressing the mRNA, allowing identification and relative abundance of each cell type. Probes that could be used include neutrophil elastase (neutrophil marker, see Fig. 6.6 ), iNOS, and arginase I (macrophage markers). If sections were labeled for these markers at 1, 3, and 5 days postwounding, a direct measure could be made on the level and maturation of the inflammatory response. In situ hybridization results could be correlated with RT-qPCR for the same results and through immunohistochemistry to detect the active protein, giving a comprehensive analysis of the healing response.


Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues

Investigation of gene expression significantly contributes to our knowledge of the regulation and function of genes in many areas of biology. In this protocol, we describe how northern blot analysis is used to identify gene expression patterns at the RNA level in human cancer cells as well as in cancerous and normal tissues. RNA molecules are separated by gel electrophoresis and are subsequently transferred to a porous membrane by capillary action. Specific sequences in the RNA are detected on the membrane by molecular hybridization with radiolabeled nucleic acid probes. Despite the development of newer methods, such as real-time PCR, nuclease protection assays and microarrays, northern blot analysis is still a standard technique used in the detection and quantification of mRNA levels because it allows a direct comparison of the mRNA abundance between samples on a single membrane. This entire northern blotting protocol takes ∼ 4 d to complete.


A Comparison of the Northern Blot Vs. Southern Blot Technique

Northern and Southern blotting are techniques used in molecular biology, which are used to detect macromolecular changes related to the DNA. BiologyWise explains the nature and principle of these techniques, and compares the differences between them.

Northern and Southern blotting are techniques used in molecular biology, which are used to detect macromolecular changes related to the DNA. BiologyWise explains the nature and principle of these techniques, and compares the differences between them.

Etymology

Southern blotting is named after Sir E. M. Southern, a British biologist who developed this technique. Subsequent blotting techniques (Northern blotting, Western blotting, etc.), which are based on the same principle, are named eponymously.

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In molecular biology and genetics, various blotting techniques are employed to detect and study changing levels of proteins, DNA, or RNA, and also to study the interactions occurring between them. Southern blotting is used in case of DNA, Northern blotting in case of RNA, Western blotting in case of proteins, and Eastern blotting in case of post-translational modifications of proteins. The purpose of each technique may be different, but all share the same principle and methodology, with a few minor deviations and modifications.

Principle

All blotting techniques are based on the same principle of specific base pairing of the probe sequences to the sequences immobilized on the membrane. Any change in the sample sequences will lead to the probes not binding or binding non-specifically. This change in binding affinity will be visualized upon secondary hybridization with a labeled probe. The intensity of the resultant dye or fluorescence will indicate the specificity of the binding along with other characteristics such as copy number, gene expression, etc.

Northern and Southern Blotting Techniques

Northern blotting is a technique used to detect and study specific RNA molecules from a mixture of different RNA, all isolated from a particular tissue or cell type. It allows the investigator to determine the molecular weight of mRNA, and also to determine the relative quantity of mRNA (gene expression) across different samples.

Southern blotting is used to detect and study specific DNA sequences. It allows one to study restricted (cut) DNA fragments, changes in the sequence, and its relative quantity across different samples.

Methodology

✦ Initially, the purified sample is loaded onto an agarose gel and electrophoresed, causing the sample to separate into bands. The bands are then transferred onto a carrier membrane by the principle of either capillary action or directed electric current. The membrane with the transferred bands is then sealed by baking in an oven or by treating it with a blocking solution. This ensures that the transfer is made permanent, and the unbound surface of the membrane is blocked so as to prevent binding of unwanted molecules and the possibility of a hampered result.

✦ Once the blocking is done, the membrane is washed gently to remove traces of the blocking solution. The membrane is then treated with specific target probes that bind to the transferred bands. The binding of the probe to the target molecule is called primary hybridization. The membrane is washed gently to eliminate the excess probes, and then it undergoes secondary hybridization, where the target probe is labeled with a radioactive element, a fluorescent dye, or even a chromogenic dye. The excess is again washed off.

✦ Depending on the type of secondary probe used, the membrane is visualized accordingly. In case of radioactive labeling or the use of a fluorescent dye, an X-ray film is exposed to the membrane for a few seconds and developed. If a chromogenic dye is used, the results can be seen on the membrane itself. Nowadays, fluorescent dyes are preferred over radioactive labeling to prevent undue exposure to radiation. However, there are some cases where one has to use radioactive labeling.

Northern Blot Vs. Southern Blot

Northern Blot Southern Blot
Development
It was developed by James Alwine, David Kemp, and George Stark in 1977. It was developed by Sir Edwin Mellor Southern in 1975.
Origin of Name
The name is a misnomer―an eponymous derivative of Southern blotting. It is named after its inventor, E. M. Southern.
Purpose
It detects presence of specific RNA sequences. It detects changes in specific DNA sequences.
Sample Preparation
The sample is used in its native state. The sample has to be denatured.
Membrane
The membrane used is an amino benzyloxymethyl filter paper membrane. The membrane used is a nitrocellulose membrane.
Type of Hybridization
RNA-DNA hybridization DNA-DNA hybridization
Application
To study gene expression profiles. To study genetic changes in DNA it can be used for homology-based cloning too.
Derivative
Northwestern blotting studies that detect interactions between RNA and proteins. Southwestern blotting studies that detect DNA-binding proteins.

The two techniques stem from the same principle and differ in the type of molecules they target. The development in the blotting techniques has helped provide scientists with a tool to study molecular interactions and detect changes, if any.

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Teaching Notes and Tips

This type of problem exposes students to a new technique in a real context, and helps them gain practice with data interpretation skills. The questions require the students to operate at a high level of thinking (synthesis and analysis) to make the connection between RNA splicing occurring in the nucleus and the bands they observe on the gel. Students often don't intuitively connect the bands on the gel with the idea of exons and introns, even though they have just heard a mini-lecture on RNA processing, including splicing and alternative splicing. They often miss the significance of the nucleus and cytoplasm labels, and need coaching to recognize that the location hints at various processes, i.e. where in the cell mRNA is synthesized and processed versus where in the cell a mature mRNA may be located and translated. As students work this problem and discuss possible answers, misconceptions are frequently uncovered for example, one common misconception is that there must be multiple genes coding for the protein and that is the reason for the multiple bands.

Note: in class we post the actual blot online. Because of copyright issues we include a representation of the blot in the student problem above.

Answer key RNA processing and northern blot technique problem


Watch the video: RNA INTERFERENCE RNAi - mechanism and applications! (August 2022).