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Polymerase chain reaction - Wikipedia

cDNA synthesis kit for first strand synthesis with random examers and then use a second …

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Animal protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Nathan Kline Institute/NYU School of Medicine and postmortem tissue accession were in full accordance with NIH guidelines. Wild-type C57BL/6 mice were euthanized by cervical dislocation and brains stored at −80°C. Cortex was weighed and approximately 100 mg was utilized for RNA extraction. Trizol (Invitrogen, Carlsbad, CA) was added at 10X (w/v) and tissue was homogenized. RNA was then extracted with chloroform and precipitated utilizing isopropanol and resuspended in 18.2 mega Ohm RNase-free water (Nanopure Diamond, Dubuque, IA). RNA purity and concentration was analyzed utilizing the total RNA Nano procedure by bioanalysis (2100, Agilent Technologies). Hippocampal CA1 neurons were harvested from postmortem human brains as described previously (). Briefly, 50 individual neurofilament-immunoreactive CA1 neurons and 50 individual neurofilament-immunoreactive dentate gyrus (DG) granule cells were microaspirated via LCM per reaction from 6 um thick paraffin embedded tissue sections (n=8 normal aged controls) and (n=6 subjects with AD) (). A total of 3–5 samples per cell type per case were collected and analyzed for both RNA amplification procedures (e.g., original TC RNA amplification protocol and TC RNA amplification without second strand synthesis).

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Array platforms consisted of 1 μg of linearized cDNA purified from plasmid preparations adhered to high-density nitrocellulose (Hybond XL, GE Healthcare, Piscataway, NJ). Each cDNA and/or expressed sequence-tagged cDNA (EST) was verified by sequence analysis and restriction digestion. cDNA clones and ESTs from mouse, rat, and human were employed. Approximately 576 cDNAs/ESTs were utilized on the current array platform. Arrays were prehybridized (2 hours) and hybridized (14–16 hours) in a solution consisting of 6X SSPE, 5X Denhardt’s solution, 50% formamide, 0.1% sodium dodecyl sulfate (SDS), and denatured salmon sperm DNA (200 μg/ml) at 42 °C in a rotisserie oven (; ). Following hybridization, arrays were washed sequentially in 2X SSC/0.1% SDS, 1X SSC/0.1% SDS and 0.5X SSC/0.1% SDS for 15 minutes each at 37°C. Arrays were placed in a phosphor screen for 24 hours and developed on a phosphor imager (GE Healthcare). All array phosphor images were adjusted to the same brightness and contrast levels for data acquisition and analysis. Hybridization signal intensity was determined utilizing ImageQuant software (GE Healthcare). Statistical procedures for custom-designed microarray analysis have been described in detail previously (, ). Briefly, the arrays were compared to negative control arrays performed utilizing the respective protocols without any starting RNA (). Expression of TC amplified RNA bound to each linearized cDNA (approximately 576 cDNAs/ESTs on the array) minus background was then expressed as a ratio of the total hybridization signal intensity of the array (a global normalization approach). Global normalization effectively minimizes variation due to differences in the specific activity of the synthesized probe as well as the absolute quantity of probe present (; ). Data analyzed in this manner does not allow the absolute quantitation of mRNA levels. However, an expression profile of relative changes in mRNA levels was generated. Relative changes in total hybridization signal intensity and percentage of cDNA clones above negative control were analyzed by one-way analysis of variance (ANOVA) with post-hoc analysis (Neumann-Keuls test; level of significance was set at p

3 Main Classification of Vectors (With Diagram)

III. Second strand Synthesis and amplification Optimisation of LD-PCR second strand synthesis / cDNA amplification (SMART PCR cDNA Synthesis Kit protocol …

In PCR one primer binds to one single stranded piece of DNA (that is why the first step is always to dentature at 94C(or so) to seperate any double stranded molecules into single strands. In the case of the first-strand cDNA from an RT reaction only one primer binds in the first round... This means that the first round of amplification in an RT-PCR is not logrithmic you simply synthesize the second strand of cDNA. In subsequent rounds (round 2 to round n) there is logrithmic amplification because both the forward and reverse primers bind to the denatured double stranded template.

In the first round, I think it is the forward primer that binds... I think this is because mRNA=forward strand in the genome and the first strand cDNA=complement of mRNA (or is the same as the reverse strand in the genome)

Someone check my logic here, I am confusing myself... is that right that the mRNA is the complement of the reverse strand of genomic DNA??
ie: mRNA sequence=forward strand=5'-3' sequence in gDNA (just with U not T)???

HTH

Terminal continuation (TC) RNA amplification was developed originally to reproducibly and inexpensively amplify RNA. The TC RNA amplification method has been improved further by obviating second strand DNA synthesis, a cost-effective protocol that takes less time to perform with fewer manipulations required for RNA amplification. Results demonstrate that TC RNA amplification without second strand synthesis does not differ from the original protocol using RNA harvested from mouse brain and from hippocampal neurons obtained via laser capture microdissection from postmortem human brains. The modified TC RNA amplification method can discriminate single cell gene expression profiles between normal control and Alzheimer’s disease hippocampal neurons indistinguishable from the original protocol. Thus, TC RNA amplification without second strand synthesis is a reproducible, time- and cost-effective method for RNA amplification from minute amounts of input RNA, and is compatible with microaspiration strategies and subsequent microarray analysis as well as quantitative real-time PCR.

On the Basis of Our Aim with Gene of Interest 2

Double strand cDNA synthesis: This page contains the detailed version of the protocol for RNA extracton and cDNA synthesis in ..

A representative comparison of the original and modified TC RNA amplification methods is presented in . Using mouse brain RNA extracts (1 ng, 10 ng, 25 ng, and 50 ng), the TC RNA amplification procedure without second strand synthesis provided robust hybridization signal intensity similar to results published previously by our group using a second strand synthesis step (). The similarity between procedures is evidenced for total hybridization signal intensity () as well as individual genes with low, moderate, and high relative expression levels including ACTB, calcium/calmodulin-dependent protein kinase II alpha (CAMK2), GRIA1, and SYP (). Hybridization signal intensity levels are depicted for representative custom-designed arrays with and without second strand synthesis in . Similar results indicated that levels of total hybridization signal intensity on the custom-designed array () as well as individual genes () did not differ statistically between TC RNA amplification procedures for human CA1 neurons and DG granule cells acquired via LCM. Additional experiments were performed to assess the fidelity of the new modification on the ability to detect expression level differences across diagnostic conditions. Specifically, microarray analysis of CA1 neurons acquired via LCM from normal control and AD brains indicated a significant down regulation of the synaptic-related marker SYP in AD with the original TC RNA amplification protocol (52.6% ± 8.6 decrease as percentage of normal aged control) or without second strand synthesis second strand synthesis (65.7% ± 11.5 decrease as percentage of normal aged control) at a similar level of high statistical significance (). Validation of the observed SYP down regulation via microarray analysis was performed with frozen human hippocampus using qPCR. Pronounced down regulation was found (82.2% ± 10.2 decrease as percentage of normal aged control), similar in direction (down regulation) and percentage of decrease to the microarray assessments with the two distinct TC RNA amplification protocols (). A greater decrease in SYP expression in AD using the qPCR technique may indicate greater sensitivity of this method for quantitation of one primer set (), or could also indicate that down regulation of SYP gene expression occurs in multiple cell types within the hippocampal formation (e.g., CA3 pyramidal neurons and dentate gyrus granule cells in addition to CA1 pyramidal neurons profiled via LCM for the present microarray analysis). Importantly, down regulation of SYP gene expression and protein levels has been observed by our group and others via several independent genomic and protein-based techniques within the AD postmortem human hippocampal formation (; ; ; ), further validating the current technical and research observations.

A new adaptation of the TC RNA amplification procedure is presented that enables robust RNA amplification without the need for second strand synthesis, a cost-saving and potential yield-preserving method compatible with homogeneous cell analysis by laser capture microdissection (LCM) and subsequent microarray analysis. Herein, TC RNA amplification without second strand synthesis is compared to the original TC RNA amplification protocol using RNA extracted from mouse brain and hippocampal CA1 neurons and dentate gyrus granule cells obtained via LCM from human postmortem brain tissues to demonstrate viability of the modification.

Addgene's guide to using CRISPR plasmids in your lab, from knockouts to pooled library screening.
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10/01/2018 · ds cDNA synthesis for RNAseq



For most cases, such for RT-PCR, 3' or 5' RACE, we just use single stranded cDNA (the first strand cDNA), while in case of preparing the cDNA for library construction, or RDA/SSH analysis which might need additional second strand synthesis step for generate double strand cDNA.

Protocol for NEBNext® Ultra™ II Directional RNA ..

A well known linear amplification method, amplified antisense RNA (aRNA) amplification (; ), enables the quantitation of relative gene expression levels from fairly small amounts of input RNA. There have been modifications of the aRNA procedure to improve efficiency (; ; ; ) and several kits that use aRNA-based technology are available commercially. The principal obstacle of problematic second strand cDNA synthesis remains. This difficulty is not exclusive for the aRNA protocol. Rather, the majority of current RNA amplification methods are afflicted by problems with second strand synthesis efficiency and specificity (; ; ). Key factors to improving RNA amplification include streamlining and/or obviating second strand cDNA synthesis and allowing for flexibility in the placement of bacteriophage transcriptional promoter sequences for sense and antisense amplification. An RNA amplification procedure developed in our laboratory named terminal continuation (TC) RNA amplification satisfies these objectives (). TC RNA amplification originally consisted of synthesizing first strand cDNA complementary to the mRNA template, subsequent second strand cDNA synthesis complementary to the first strand cDNA, and finally IVT using the double stranded cDNA as template (). First strand cDNA synthesis complementary to the template mRNA entails the use of two oligonucleotide primers, a first strand poly d(T) primer and a TC primer () (). Transcript orientation with TC RNA amplification procedure can be in the antisense orientation when the bacteriophage promoter sequence is placed on the first strand poly d(T) primer or in a sense orientation when the promoter sequence is attached to the TC primer. One round of amplification is sufficient for downstream genetic analyses (). TC RNA amplification has been shown to be more sensitive than aRNA amplification using input RNA obtained from neurons from mouse and postmortem human brains (; ).

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