RNA Probe

Methods to probe RNA secondary structure, such as small molecule modifying agents, secondary construction-specific nucleases, inline probing, and SHAPE chemistry, are widely used to study the structure of functional RNA.

From: Methods in Enzymology , 2015

Molecular Biology and Genomics : A Primer for Neurosurgeons

H. Richard Winn Medico , in Youmans and Winn Neurological Surgery , 2017

The Candidate Gene Approach

Lacking any biochemical ground that would aid in purifying gene products associated with a disease state, the candidate gene arroyo was largely an outgrowth of the efforts of positional cloning strategies in the early 1980s. Using linkage analysis to await at differences in chromosome construction in diseased versus nondiseased individuals often in tandem with linkage disequilibrium mapping to define broad (i.e., <10 centimorgans) and much finer respective chromosomal intervals associated with a priori knowledge of their etiologic office in a affliction state enabled molecular geneticists to narrow the number of candidate causative genes. Before the completion of the Human Genome Project, identifying these loci was an incredibly arduous and time-consuming task, in large office because of the lack of high-resolution genetic and physical chromosomal maps. The unprecedented efforts to surmount these struggles were most famously recorded in the midst of the pioneering work of Louis Kunkel and Ronald Worton in identifying dystrophin equally the factor responsible for Duchenne's muscular dystrophy. 1 In the by several decades, numerous high–relative-chance genes associated with similar "simple" mendelian diseases have been successfully identified with these methods. More 30% of mendelian disease has neurological manifestations. 2 Past applying the tools of molecular biology to a gene or the pocket-size set of candidate genes identified within a quantitative trait loci, it has became possible to facilitate a systematic approach to answering some very bones questions about the organization of a factor and the expression of its factor products.

Expanding on the sequence of methods conceptualized starting time past Sol Spiegelman iii and near effectively by Edwin Southern, iv molecular biologists accept exploited the now well-worn principle of molecular hybridization in which a single-stranded nucleic acid probe (or primer) forms a stable hybrid molecule, as a effect of nucleotide complementarity, with a single-stranded target sequence immobilized on a solid back up (i.east., nitrocellulose or nylon membranes) or in solution (Fig. 44-i). Nether the appropriate experimental conditions the stability and biochemical kinetics of the hybrid are straight proportional to the length and the degree of nucleotide complementarity.

Equally starting time practical in Southern blotting whereby genomic Dna was size-separated using agarose gel electrophoresis, transferred to nitrocellulose, and annealed to complementary Deoxyribonucleic acid probes labeled with a detectable tag, the dosage or deletion analysis, or both, of candidate genes was affirmed. Indeed, Southern absorb analysis of the dystrophin gene has identified duplications as well as mapped various exon deletion mutants. Recently, variation in the copy number of genes has received new consideration as several new genomic disorders have been shown to manifest in a gene dosage-dependent manner. 5 These include dup7 (q11.23) syndrome, MECP2, and adult-onset autosomal ascendant leukodystrophy. Past reducing the stringency of hybridization conditions, still, differences in hybridization patterns may reveal the existence of sure fragments that are not able to hybridize to the probe under the nearly stringent hybridization atmospheric condition. These data are frequently the first clues that the gene is office of a larger multigene family that shares significant merely not complete nucleotide sequence identity. By using a modification of the Southern blotting method to screen a complementary Dna library at moderate stringency, the dystrophin-similar sequence utrophin was identified. half dozen

NUCLEIC ACIDS | Immunoassays

J.M. WagesJr, in Encyclopedia of Analytical Science (2d Edition), 2005

RNA probes

RNA probes bind tighter to their complementary strands than do Deoxyribonucleic acid probes. Poor stability due to ubiquitous ribonucleases has hampered more widespread use of short RNA probes, as has the difficulty of efficient chemic synthesis of long RNA oligomers. Contempo advances in RNA synthetic chemical science take solved the latter problem.

Long RNA probes are generated by in vitro transcription from linearized plasmid Dna containing a promoter sequence for a DNA-dependent RNA polymerase such as SP3, T3, or T7 polymerases. Commercially available kits for high-yield transcription with label incorporation are available. Information technology is besides technically trivial to innovate a T7 promoter sequence every bit a v′-extension of a PCR primer. Distension introduces the promoter sequence into the amplicon. Transcription of the amplicon with T7 RNA polymerase and a modified NTP yields the labeled RNA probe. Shorter RNA probes (less than ∼50 bases) are chemically synthesized, and characterization is most conveniently introduced during synthesis.

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Clinical Neurogenetics

Joseph Jankovic Dr. , in Bradley and Daroff's Neurology in Clinical Practice , 2022

Hereafter Function of Systems Biology in Neurogenetic Disease

The complex relationship between genetic risk variants, even when they are inherited in a Mendelian fashion, and clinical features, or the human relationship of these mutations to disease pathophysiology, nowadays significant challenges to the use of genetics for diagnosis and therapeutics. There are many diseases, including almost all neurodegenerative disorders, where cognition of the specific causative cistron has non immediately yielded new curative therapies but has instead raised many new questions regarding the underlying molecular etiology of the disease. Although there is promise for development of gene-specific therapies in the near future for a small percentage of these conditions (Rexach et al., 2019), for the residue, research into the underlying mechanisms is essential to uncover new therapeutic targets. Toward that goal, the technologies discussed have fabricated greater amounts of information available for scientific analysis than ever earlier. For example, microarrays can be used to study not simply genome-wide genetic variation via SNPs, every bit described earlier, merely likewise variations in gene expression (Fig. 48.14). For this method, the array platform contains probes that are complementary to genome-wide mRNA sequences, and the report is performed by hybridizing the assortment with fluorescently labeled mRNA collected from either patients or controls. The intensity of the fluorescent signal can be used to determine and compare the relative levels of expression for each gene across the samples. Like techniques tin too be used to evaluate RNA splicing with probes that correspond to all the exons in a given gene then assessing samples for their alternative usage in cases and controls. Next-generation sequencing can also exist used to study RNA expression and splicing on a genome-wide scale in private tissues or even in single cells (Gandal et al., 2018; Wang et al., 2018). With the availability of data encompassing both genetic variation and gene expression in clinically evaluated patients and controls, it becomes possible to contain and synthesize the totality of this information together in ways that appraise phenotype, genetic variation, and gene expression simultaneously in a more than comprehensive way. This subject area, known equallysystems biology, strives to utilise these sets of information to develop detailed genetic pathways to identify related genes and genetic programs relevant to disease (Geschwind and Konopka, 2009; seeFig. 48.14). Such integrative assay, a critical aspect to the concept of precision wellness (Ashley, 2015; Rexach et al., 2019), has begun to accelerate our understanding of disease pathogenesis and generate new insights into more than constructive treatment strategies, which will but improve every bit nosotros larn more and the techniques improve.

One example of this blazon of systems biological science arroyo involves using cistron expression data, such as from microarray studies, to grouping private genes co-ordinate to their degree of coexpression, forming functionally related cistron expression modules. These modules are then graphed co-ordinate to the interconnectivity of their members, which produces a network of correlations centered around one or more than key genes, termedhubs, which functionally bulldoze the association either directly or indirectly. Farther assessment of these hub genes and their connections tin can identify potentially important genes and biological pathways affected in affliction. Such techniques have been applied to the report of Advert (Miller et al., 2008, 2010). Epilepsy (Winden et al., 2011), HIV-associated dementia (Levine et al., 2013), ALS (Saris et al., 2009), chronic fatigue syndrome (Presson et al., 2008), hereditary cerebellar ataxia (Fogel et al., 2014b), and schizophrenia (Torkamani et al., 2010), and take already led to new therapeutic targets (Swarup et al., 2019). In one of these examples, the observance of shared molecular disease underpinnings betwixt humans and mice with drastically differing phenotypes (Fogel et al., 2014b) led to the observation of a new phenotype in patients (Becherel et al., 2019). These various systems biology studies illustrate the versatility of such an approach and the potential bear on these studies can have on research into complex illness pathogenesis.

Methods for Nonradioactive Labeling of Nucleic Acids

Christoph Kessler , in Nonisotopic Probing, Blotting, and Sequencing (2d Edition), 1995

B RNA Labeling Techniques

RNA probes are ordinarily synthesized with the RNA polymerases from bacteriophages SP6, T7, or T3 by in vitro transcription of Deoxyribonucleic acid, which is cloned into appropriate transcription vectors like the pSPT-vector family containing highly specific SP6-, T7-, or T3-specific promoters in front of a multiple cloning site (Melton et al., 1984; Morris et al., 1986; Krieg and Melton, 1987). Cloning particular Dna fragments in one of these restriction sites results in consummate transcription units acting as templates for DNA-dependent RNA synthesis. The reaction scheme of in vitro RNA synthesis is shown in Fig. 4.

Fig. iv. "Run-off" RNA synthesis.

The RNA polymerase-catalyzed reaction usually starts by initiation of the transcription reaction with a purine ribonucleoside triphosphate at a fixed position downstream of the promoter sequence (Butler and Chamberlin, 1982). If a modified ribonucleotide is added to the nonmodified nucleotide mixture, the growing RNA chain is labeled by integration of the modified ribonucleotide. Mostly hapten-labeled CTP or UTP (e.chiliad., bio-CTP; DIG-UTP) is used as labeling reagent (Theissen et al., 1989; Höltke and Kessler, 1990; Höltke et al., 1992b; Rashtchian and Mackey, 1992). The transcription reaction is terminated at the cease of the linearized transcription unit; thus hapten-modified probe molecules of divers length and sequence are synthesized. The completion of the transcription reaction requires ane-2 hr depending on the desired labeling density; east.chiliad., with digoxigenin, labeling with 35% digoxigenin-modified UTP and 65% UTP reaches an optimum afterward 2 hr with T7 RNA polymerase. Using I μg template DNA and I mGrand ATP, GTP and CTP each, 0.65 mM UTP and 0.35 gM DIG-UTP in 20 μl transcription buffer including 20 units RNase inhibitor, up to 20 μ-thousand labeled transcripts may be obtained. Thus, in vitro labeling with RNA polymerases may exist used for preparative RNA probe synthesis.

Subsequently the transcription reaction, the template Dna tin can be removed by digestion with RNase-costless DNase. The resulting labeled RNA probe contains no vector sequences, so that cantankerous-hybridization caused by unspecific interaction between vector and target sequences is absent during hybrid formation.

Although the cadre region of phage-specific promoters is only 17 nucleotides in length and differs in only a few nucleotides between SP6, T7, and T3 promoters (Pfeiffer and Gilbert, 1988), the promoters are highly specific for the respective RNA polymerase (Butler and Chamberlin, 1982; Kassa- vetis et al., 1982). After linearization of the recombinant clone direct downstream of the transcription unit of measurement, run-off transcripts of defined length are synthesized. By positioning two promoters of dissimilar specificity and polarity at both sides of the transcription unit, transcription of both sense (coding) and antisense (noncoding) RNA can be initiated.

The advantages of RNA probes (e.chiliad., single-strandedness, not-selfcomplementarity, defined length, and higher stability of the RNA/Deoxyribonucleic acid hybrids have been widely exploited in different hybridization applications. In virtually cases radioactively labeled probes have been used and these accept all the pitfalls of instability, low resolution, and all the disadvantages of handling isotopes. Applying nonradioactively labeled RNA probes in blot experiments as little every bit 0.1 pg homologous DNA or RNA can be detected in dot, Southern or Northern blots. This sensitivity is comparable with that obtained with radioactively labeled RNA probes.

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Diagnosis of Human being Immunodeficiency Virus Infection

John E. Bennett MD , in Mandell, Douglas, and Bennett's Principles and Practise of Infectious Diseases , 2020

Hybridization and Amplification Assays to Observe HIV Nucleic Acids

Use of nucleic acid testing (NAT) represents an adjunct to, just not a replacement for, serologic methods for HIV detection. Various formats can detect HIV-ane RNA with great sensitivity, and new real-time polymerase chain reaction (PCR) methods accept been developed capable of detecting a single copy of HIV-1 RNA. 195 Clinical methods for HIV nucleic acid detection for use in monitoring HIV infection have included nucleic acid sequence-based amplification (NASBA), branched Dna (bDNA), and PCR amplification; NASBA formats accept detected equally depression every bit 22 to 31 copies/mL. 196,197 The get-go of these methods to exist FDA canonical for donor screening or diagnosis is a "TMA/HPA" system (Fig. 120.half-dozen) consisting of a transcription-mediated distension (TMA) and hybridization protection assay (HPA), named Aptima, developed by Gen-Probe (San Diego, CA). 54 This modality has been approvedboth for screening and confirmation of infection and but detects HIV-one.

The principle of the TMA/HPA assay is to generate large numbers of HIV RNA copies from HIV-i for detection past specific hybridization to chemiluminescent probes (run acrossFig. 120.6). Plasma samples are extracted, and HIV-1 RNA is reverse transcribed to complementary Dna (cDNA) using exogenous murine leukemia virus reverse transcriptase. The primer for the cDNA reaction contains the promoter sequence for the T7 bacteriophage RNA polymerase, followed by sequences complementary to HIV-ane. The resulting cDNA product contains T7 promoter sequences linked to HIV-i sequences, which are used every bit a template for added T7 RNA polymerase, a high-efficiency enzyme that chop-chop transcribes multiple copies from the chimeric T7-HIV cDNA. The RNA copies are visualized by addition of chemiluminescent probes, which hybridize to HIV-ane sequences; excess probe is quenched; and the luminescence from the RNA-probe hybrid is quantitated (seeFig. 120.6). The assay is qualitative but and does non yield specific re-create number; sensitivity of the analysis is in the range of 13 copies/mL plasma. 54 TMA/HPA detected a express panel of non-B subtypes of HIV-1 equally well. The TMA/HPA analysis was sufficiently sensitive to detect HIV RNA 12 days earlier than standard third-generation ELISA and vi days before than p24 assays. Thus one primary application of TMA/HPA is in diagnosis of early HIV-1 infection; the Aptima system was approved for screening and confirmation of HIV-one in 2006 and should replace use of bDNA or PCR assays for diagnosis.

Similarly, TMA/HPA technology was developed for blood donor screening. In the United States trials for claret donor screening by NAT screened more than than xx million donations and found 7 HIV-infected samples that scored negative for HIV antibody. 102 NAT systems detected a number of HIV-1 infections that would take been missed past previously licensed test methods, confirming the increased sensitivity of these systems. 198 Similarly, trials in Europe, South Africa, and Japan 199–202 and in case reports 203–207 have documented, either in existent time or retrospectively, identification of patients in seroconversion windows, using NAT, who had scored negative using p24 antigen detection. Past contrast, at that place are as well reports of undetected HIV-one in the setting of relatively depression HIV-ane RNA levels, peculiarly in minipools of 16 or 24 plasma samples, 102 Retrospective analysis of a blood manual case (before NAT) revealed a relatively depression viral RNA level (estimated twoscore copies/mL) 208 ; the benefit of NAT over traditional testing has been estimated to reduce the window period by two to 6 days. Using NAT applied science, the chance of HIV infection through transfusion was estimated as 1 per 1,576,000 209 ; four cases of p24 antigen negative blood component were identified in more than xix,000,000 screened units. 210 In upshot, the take a chance of HIV-1 transmission via blood components is reduced, merely not completely eliminated, by incorporating NAT into screening procedures; the residual take chances of HIV infection in United states is estimated at 1 per two,135,000. 210 Like or lower risks have been estimated from other reporting countries, 211,212 and although there are concerns regarding subtype sensitivity and sample preparation, many blood centers throughout the world have incorporated NAT as a component of HIV screening. The relative cost-effectiveness of NAT remains a business organisation. 199,201,213 NAT is canonical in the Usa for blood-donor screening using plasma, and assays must be able to find 100 copies 95% of the time; several assays exceed this limit. 197 Standard subtype B virus preparations have been established as quantitative controls. 214 Testing tin can exist performed on single samples or minipools of plasma and may be combined with testing for hepatitis C and B.

Gene Probes

C. Anthony Altar , ... James H. Eberwine , in Methods in Neurosciences, 1989

RNA Probes

RNA probes ( Angerer and Angerer, 1981) are made past in vitro transcription of template DNA by an RNA polymerase which initiates RNA synthesis at specific binding sites called promoters (see Fig. 6). There are several commercially available plasmid vectors with the SP6, T3, or T7 promoters separated by a multiple cloning site (eastward.g., Promega's pGEM-iii plasmid) into which the DNA of interest is cloned. The protocol to be described is by Angerer et al. (1987). The plasmid template (0.2–0.5 mg/ml) is linearized past a restriction endonuclease that cuts once downstream of the insert sequence, just not elsewhere between the insert and promoter. The buffer is removed by dialysis of the Dna at room temperature for 30 min against v mM Tris, pH 8.0, past placing 25–100 μl of the sample on the shiny hydrophobic side of a VMWP small-scale-pore nitrocellulose filter (Millipore) on top of the dialyzate. The reaction is prepare as follows: (1) dry the radioactively labeled ribonucleotides in a microcentrifuge tube nether vacuum (for a 10–100 μM last concentration); (2) add xx μl of a solution containing forty kK Tris, pH seven.5,6 mM MgCl2, ten mM DTT, 100 μM unlabeled NTPs, 2 mM spermidine, and 100 U/ml placental RNase inhibitor (Promega Biotech); (three) add the template DNA (100 µg/ml) to the tube, mix rapidly (to avoid precipitation of the Dna by the spermidine); (4) add 1200–1800 U/ml of the appropriate RNA polymerase. Incubate the reaction at 37 to 41°C for lx min.

Fig. half dozen. Schematic of the kinasing, 3′-tailing, and RNA probe labeling of ISH oligonucleotide probes. Come across the text for further details.

Measure incorporation of the nucleotides past TCA precipitation. The probe is purified by removing the Deoxyribonucleic acid template with a 30-min digestion at 37°C with RNase-complimentary DNase I (50 µg/ml in 50 kM Tris, pH 7.4,10 grandM MgCl2). The probe is and so extracted with an equal volume of phenol/chloroform and ethanol precipitated with 0.2 M ammonium acetate and 2.5 vol of ethanol.

RNA probes accept the advantage of using known "sense" and "antisense" strands for hybridization to the mRNA of interest and for the command of background hybridization. The "antisense" sequence is complementary to the RNA sequence and will hybridize to the mRNA. The "sense" sequence is the same as the mRNA. Either probe is single-stranded, and then reannealing of the probe in solution is not a problem, and thus they can exist labeled to high specific activity. I disadvantage is that RNA is very sensitive to degradation past RNase, and so precautions must exist taken to avoid degradation, such equally autoclaving all the solutions and wearing gloves. Another problem is that RNA probes are "sticky," in that they adhere to more than than just their complementary RNA. These nonspecific associations are lessened by treating sections with RNase following ISH. This reaction should exist done in 0.v K NaCl to prevent the RNase from digesting the RNA–RNA hybrids.

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Retinoid Signaling Pathways

Stephen R. Shannon , ... Paul A. Trainor , in Methods in Enzymology, 2020

vi.2 Protocols

RNA probe synthesis

i.

Mix the following at room temperature:

Sterile distilled water (DEPC-H2O) 11.5   μL
5   × transcription buffer 5.0   μL
100   mM DTT 2.5   μL
DIG 10   × nucleotide mix 2.5   μL
Linearized plasmid (one   μg/μL) 1.5   μL
Rnasin ribonuclease inhibitor 0.v   μL
Polymerase (SP6, T3 or T7) 1.five   μL
Full 25   μL
2.

Incubate at 37   °C for 2   h up to 6   h.

3.

Remove 1   μL aliquot and run on i% TAE gel to check synthesis. Expect to run across RNA band 10-fold more intense than plasmid band, suggesting 10–15   μg probe.

4.

Add together 2   μL of DNAse1 (ribonuclease free) and incubate at 37   °C for 15   min.

5.

Add:

50   μL dH2O
25   μL 10   Grand ammonium acetate
200   μL 100% ethanol
6.

Mix and exit on dry ice for 30   min or store at −   80   °C overnight.

7.

Spin in centrifuge at 4   °C for xx   min at xiii,000 to xv,000   rpm.

8.

Wash pellet in 50   μL of lxx% ethanol and spin at 4   °C for five   min at xiii,000 to 15,000   rpm.

ix.

Air dry pellet for 5–10   min until obvious traces of ethanol have evaporated.

10.

Redissolve pellet in 50   μL hybridization solution and store at −   20   °C. If a college concentration of probe is needed the pellet may be resuspended in 30   μL of hybridization solution.

In situ hybridization staining method

1.

Dissect embryos in DEPC-PBS and fix in 4% paraformaldehyde for 2   h to overnight (use scintillation vials).

2.

Wash embryos in PBT at room temperature for x   min.

iii.

Dehydrate the embryos through a graded serial of methanol diluted in PBT: wash in 25% methanol, 50% methanol, 75% methanol and 100% methanol for 10   min each.

4.

Embryos tin can be stored at −   20   °C up to several weeks at this stage.

5.

Rehydrate the embryos through a graded series of methanol diluted in PBT: wash in 75% methanol, 50% methanol, 25% methanol for ten   min each.

vi.

Wash in PBT for 5   min at room temperature.

7.

Incubate in 10   μg/mL proteinase K (diluted in PBT) for 5–10   min at room temperature. Incubation time will vary depending on the embryo's stage.

Incubation time by embryo stage:
E6.5: 4   min
E7.5: 4–5   min
E8.5: 6   min
E9.v: x   min
E10.5: 15   min
eight.

Cease the proteinase K reaction by washing with 2   mg/mL glycine (diluted in PBT) for five   min at room temperature (make the same day of use).

9.

Wash for 5   min in PBT at room temperature.

10.

Re-fix the embryos in iv% paraformaldehyde/0.25% glutaraldehyde for xx   min at 4   °C.

xi.

Launder for 10   min in PBT at room temperature.

12.

Transfer the embryos to 2   mL Eppendorf tube with a round bottom (up to 10 embryos can be processed in a single tube).

xiii.

Incubate embryos for at least i.5   h in 1–two   mL of pre-warmed hybridization buffer at 62   °C to 70   °C.

xiv.

Incubate embryos with 2 μL of RNA probe per 1  mL of hybridization buffer at 62   °C to 70   °C overnight in hybridization oven (ane   mL of buffer is enough for 10 mouse embryos at E10.5).

fifteen.

Wash embryos twice in 2   mL of 2xSSC/0.1% chaps for 45   min at 62   °C.

16.

Launder embryos in ii   mL of 0.2xSSC/0.1% chaps for 30   min at 62   °C.

17.

Wash in KTBT for five   min at room temperature.

18.

Incubate the embryos in 2   mL of 20% goat serum (or lamb serum) in Potassium-Tris Buffer with Triton (KTBT) for at to the lowest degree i to 1.5   h at room temperature with continual rocking.

19.

Replace blocking solution with fresh 20% goat serum in KTBT. Add together ii   μL of DIG-alkaline phosphatase antibody and incubate at 4   °C overnight with continual rocking.

twenty.

Rinse the embryos at to the lowest degree twice in KTBT.

21.

Launder the embryos between iv and 5 times in KTBT for 1   h at room temperature with continual rocking.

22.

Replace with ii   mL of fresh KTBT and launder overnight at 4   °C with continual rocking.

23.

In the forenoon, launder the embryos at least twice with KTBT for a combined time of 30   min at 4   °C.

24.

Wash the embryos in ane   mL of alkaline phosphatase buffer for 10   min at room temperature with continual rocking.

25.

Add together iii.375   μL of NBT (100   mg/mL in DMF) and iii.v   μL of BCIP (50   mg/mL in DMF) directly to the embryos in alkaline metal phosphatase buffer with shaking at room temperature in the dark (wrap the Eppendorf tube in foil or place in a covered container).

26.

Stop the reaction once the desired colour intensity is achieved past fixing the embryos in four% paraformaldehyde. This can take from xv   min to overnight, however i to 2   h is more common. Continual ascertainment of the colour development is not advisable as the background volition increase.

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Nucleic Acid Probe Technology

Robert E. FarrellJr. Ph.D , in RNA Methodologies (Fourth Edition), 2010

Characteristics of RNA probes

1.

RNA probes are single-stranded. While such probes do not require boiling prior to use, heating them briefly will help to adjy any intramolecular base-pairing that may take occurred.

2.

All RNA probe molecules are available for hybridization. Single-stranded, denatured RNA molecules cannot renature, as do dsDNA, although some intramolecular base-pairing, should it occur, may reduce the effective concentration of the probe.

3.

RNA probes are continuously labeled as they are being transcribed, thereby generating probes with a very loftier caste of label incorporation.

four.

RNA probes testify greater thermodynamic stability when base-paired with either DNA or RNA target molecules, compared to the thermodynamic stability associated with Dna probes.

5.

RNA probes are synthesized by in vitro transcription from a linearized template; therefore, all probe molecules are of uniform length.

6.

Enormous quantities of probe tin can exist synthesized in a single in vitro transcription reaction ix .

7.

The SP6, T7, and T3 bacteriophage RNA polymerase promoters demonstrate near no cross-reactivity; therefore, transcription reactions initiated from one promoter or the other are virtually free of transcripts of the opposite sense. Consequently, both sense and anti-sense RNA probes can be synthesized every bit needed.

8.

RNA transcribed in vitro, as with all RNA, must be treated with RNase-free reagents; failure to do and then will result in rapid degradation of the probe.

9.

RNA probes ofttimes produce unacceptable, high levels of background; thus, at the conclusion of the hybridization catamenia it is a common practice to digest all probe molecules with RNase A and RNase T1. This treatment volition outcome in deposition of all probe molecules that did non participate in duplex formation.

10.

Radiolabeled RNA probes are often of such high specific activity that they may experience radiolysis if stored for extended periods.

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Nucleic Acid Probe Technology

Robert Eastward. FarrellJr. Ph.D. , in RNA Methodologies (Tertiary Edition), 2005

Characteristics of RNA Probes

1.

RNA probes are single stranded. Although such probes do not crave boiling before employ, heating them briefly will disturb intramolecular base pairing that may have occurred.

2.

All RNA probe molecules are bachelor for hybridization. Considering they are single-stranded, they cannot renature as in the case of Deoxyribonucleic acid, although intramolecular base pairing, should information technology occur, may reduce the effective concentration of the probe.

iii.

RNA probes are continuously labeled as they are being transcribed, thereby generating probes with a high degree of label incorporation.

four.

RNA probes show greater thermodynamic stability with both DNA and RNA target molecules, compared to the thermodynamic stability associated with DNA probes.

5.

RNA probes are synthesized by in vitro transcription in a template-dependent fashion; therefore, all probe molecules are of uniform length.

half dozen.

Large quantities of probe tin can be synthesized in a unmarried, in vitro transcription reaction. ix

7.

In the structure of the transcription template, the cDNA to be transcribed is generally flanked by highly efficient bacteriophage RNA polymerase promoters. The more than useful vectors contain dual RNA polymerase promoters in opposite orientations flanking a multiple cloning site. The most mutual constructions characteristic SP6 and T7 or T3 RNA polymerase promoters. Thus, both sense and antisense RNA probes can exist synthesized every bit needed.

eight.

The SP6, T7, and T3 bacteriophage RNA polymerase promoters demonstrate about no cross reactivity; therefore, transcription reactions initiated from one promoter or the other are virtually free of transcripts of the opposite sense.

9.

Plasmids used for RNA probe synthesis must exist linearized before initiating in vitro transcription.

10.

RNA transcribed in vitro, equally with all RNA, must be treated with RNase-free reagents; failure to practise so will event in rapid deposition of the probe.

11.

RNA probes frequently produce unacceptable, high levels of background; thus, at the conclusion of the hybridization period, it is a common practice to digest all probe molecules with RNase A and RNase T1. This treatment will result in degradation of all probe molecules that did non participate in duplex formation.

12.

Radiolabeled RNA probes are often of such high SA that they may experience radiolysis if stored for extended periods.

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Neuropeptide Technology

Joseph A. Majzoub , Gail 1000. Adler , in Methods in Neurosciences, 1991

Generation and Isolation of the 32P-Labeled cRNA Probe

The labeled cRNA probe is generated using [α-32P]UTP as described in the previous section (13). In the RNase protection analysis information technology is very important that the cRNA probe be full-length. To achieve this ane should try to limit the size of the probe to under 600 nucleotides. If the labeling reaction is non yielding full-length probe, the amount of [32P]UTP may be limiting, and an equal concentration of unlabeled UTP can exist added.

After eluting the labeled cRNA probe from the Elutip column in 300 μl of high-salt buffer, it is precipitated by the addition of 25 μg of tRNA and ii.five volumes (750 μl) 95% ethanol. The mixture is vortexed, frozen at −lxxx°C for xxx min, centrifuged at iv°C for 30 min, and the pellet washed with 125 μl of 70% ethanol earlier drying. The cRNA probe is resuspended in 4 μl formamide sample dye, denatured at 85°C for iii min, and electrophoresed on a 6% acrylamide gel at approximately 1500 V. The gel temperature should be around l°C. The full-length probe is excised, using an autoradiograph of the gel every bit a guide, and the gel piece is incubated in 500 μl Folio extraction buffer for 1 hour at 37°C. Twenty-five micrograms of tRNA is added, and the cRNA probe is ethanol-precipitated. The dry probe is resuspended in water. Information technology is best to use the probe the same day it is prepared.

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