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RNA extraction, Quality Assessment, and cDNA synthesis

Synthesis of good quality double-stranded cDNA from …

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can be used for routine quality control assessment of cDNA synthesis

Since first-strand cDNA synthesis is less than 100% efficient, I doubt whether it can make the smear many times brighter than the mRNA in the original total RNA.)

2) Will there be a difference in result if an oligo(dT) or anchored oligo(dT) primer is used versus a random hexamer primer?

if you start with 1 μg of total RNA for cDNA synthesis and use 1/10th of the ..

The integrity of the total RNA used for synthesizing double stranded cDNA was assessed by amplifying a 1282 bp product targeting the glyceraldehyde 3-phospho dehydrogenase (GAPDH) gene by PCR.

were used to calculate the total RNA input for cDNA synthesis.

whenever used for cDNA synthesis, was purified from total RNA using Qiagen Oligotex mRNA Maxi Kit

High quality cDNA synthesis for downstream qPCR analysis is essential for successful expression studies. Many factors affect cDNA synthesis including the reverse transcripase, buffer systems, enhancers and priming strategy. The PCRBIO cDNA synthesis mix removes the need for user optimisation of these critical factors.

The RTase is not inhibited by ribosomal and transfer RNAs, total RNA is an ideal substrate. The 5x cDNA synthesis mix can be used with 4.0pg to 0.4μg total RNA or oligo(dT) purified mRNA. The relative concentrations of random hexamers and anchored oligo(dT) have been optimised for the generation of cDNA for use in real-time PCR experiments.

How can I analyse the quality of my cDNA? - …

Has been developed to generate high-quality first-strand cDNA from total RNA template

We used the vector-primer method because it should be very efficient from a theoretical view, and our data support this. It differs from the Okayma's method (,) principally by ligation of an oligonucleotide containing the dT ‘tail’ to the vector instead of using terminal transferase to add an oligo(dT) ‘tail’ to the vector, and by a simple self-ligation reaction to circularize the product instead of a second terminal transferase reaction, enzyme digestion and ligation of a joining fragment. Thus, it is much simpler to perform. A procedure very similar to the one we used has been previously described (,), but its use was limited by the presence of an unidentified exonuclease activity; it was not applied to total RNA, and libraries produced with it were not analyzed by sequencing. We used pBluescript as the scaffold for this procedure, but it should be equally applicable to almost any cloning vector.

We have established a method that can produce complex, high-quality cDNA libraries from microgram quantities of total RNA. If a random sequencing strategy is used, the number of clones produced is many more than can be analyzed. The amount of starting material required is less than other methods that do not use an amplification procedure such as PCR. We started with 5 μg of total RNA. Purification of polyadenylated RNA from total RNA typically yields 1% of the input mass (). Thus, our method used the equivalent of ~50 ng of poly(A)+ RNA to produce several hundred thousand independent recombinants, and less starting material could probably be used to make a satisfactory library. While the number of clones sequenced from each library may limit the conclusions that can be drawn, the analysis that we have been able to perform, suggests that the libraries are both representational and diverse. The thymus and adrenal libraries, which were made from the most complex tissues, had >90% unique (i.e. different, single-copy) clones. In the pituitary library, which was prepared from a tissue with a relatively small number of highly specialized cell types, 55% of the clones were unique and the redundant clones represent abundant, organ-specific transcripts, as one would expect from a specialized secretory organ. Thus, the size and complexity of the libraries suggests that they are likely to be useful for a variety of applications that require good representation of the entire repertoire of genes expressed in a tissue.

the Transcriptor High Fidelity cDNA Synthesis Kit from Roche produced the highest yield and the best quality of cDNA.
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  • cDNA Synthesis & RT-PCR | NEB

    Your purified total RNA ..

  • SensiFAST™ cDNA Synthesis Kit - Bioline

    07/08/2009 · Each cDNA synthesis reaction can accommodate up to 5 μg of RNA

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cDNA/Reverse Transcriptase Tips | NEB

cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5–5 × 105 primary transformants, starting with 5 μg of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of ~1000 clones sequenced, ~20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.

RNA & cDNA Synthesis Services - GCC Biotech (I) Pvt. Ltd.

Total RNA or mRNA can be used in the reverse transcription reaction. Total RNA is generally sufficient for cDNA synthesis reactions. However, if desired, mRNA can be easily obtained using a polyA Spin mRNA Isolation Kit (NEB ) or Magnetic mRNA Isolation Kit (NEB ).

Total RNA, Human Internal Organ - Clontech

- High-quality full-length-enriched amplified cDNA for various applications including next generation sequencing (Roche 454, Illumina Solexa, ABI SOLiD)
- From limited amounts of total or poly(A)+ RNA

Pesquise com mais rapidez e eficiência aqui! Cdna Synthesis

Since first-strand cDNA synthesis is less than 100% efficient, I doubt whether it can make the smear many times brighter than the mRNA in the original total RNA.)

The intensity of the smear depends on how much you load.

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