Sensitive first-strand cDNA synthesis of RNA ..
The ProtoScript First Strand cDNA Synthesis Kit is contains everything needed for cDNA synthesis
Contact Quantabio for your qScript microRNA cDNA Synthesis Kit ..
We next examined whether chum-RNA would allow efficient construction of a cDNA library from a single-cell amount of mRNA. Hereafter, we will call this a single-cell cDNA library, even though we obtained the single-cell mRNA quantity by dilution of the original stock obtained from a million-cell quantity of mRNA. According to the linker-primer method (), cDNA was synthesized from a single-cell amount of 293T mRNA, converted into double strand DNA, and an adaptor added. The DNA was digested with NotI, the unnecessary portion removed by column chromatography and mRNA was amplified using T7 RNA polymerase in the presence of chum-RNA, as described above (B). Since this single round of mRNA amplification did not produce an amount of mRNA sufficient to make a good cDNA library harboring a high number of independent clones (complexity), the amplification procedure was repeated several times. Four rounds of mRNA amplification in the presence of chum-RNA produced a high-quality cDNA library containing independent cDNA clones of 6.6 × 105 colony forming unit (cfu), from only a single-cell amount of 293T mRNA. In contrast, omission of chum-RNA resulted in a very poor-quality cDNA library with only background quantities of independent cDNA clones (5.3 × 104 cfu) and no evidence that any plasmid clones carried human cDNA inserts (data not shown).
The efficiency of cDNA synthesis in the presence or absence of chum-RNA was confirmed by RT–PCR of Homo sapiens glyceraldehyde 3-phosphate dehydrogenase (HsGAPDH) mRNA using the following primer set which detects a band 902 base pairs (bp) in size; forward (HsGAPDH-F) 5′-CGA GAT CCC TCC AAA ATC AA-3′ and reverse (HsGAPDH-R) 5′-AGG GGT CTA CAT GGC AAC TG-3′. The annealing temperature for PCR was always at 50 or 55°C, while the number of amplification cycles was 30, 40 or 50.
Synthesize full-length cDNA from ng of total RNA
Acquisition of high quality, intact RNA, free of genomic DNA and RNase traces, is vital for the synthesis of a full-length cDNA followed by an accurate quantitative analysis (qPCR). The following recommendations for working with RNA should therefore be followed:
Attenuator (French : atténuateur) A sequence of bases that occurs in the leader sequence of some operons and controls transcription. Synthesis of RNA may be terminated at this site.
Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA ..
Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 × 105 cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (Km) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.
Comparison of gene-expression patterns between cells and/or tissues facilitates the identification of molecules activated by a particular physiological or pharmacological treatment. The use of gene-expression profiling is particularly important in neuroscience, clinical science, stem cell biology, and metagenomic analysis. In many cases, however, the amount of specimen tissue available is limited, allowing only small amounts of mRNA to be obtained. As such, amplification of the isolated RNA is obligatory to obtain the microgram amounts of RNA required for microarray analysis or cDNA library preparation. Without amplification, such amounts of RNA would be obtainable only from millions of cells. Although the polymerase chain reaction (PCR) is a powerful method for amplifying a single target DNA, the exponential amplification that can be achieved using multiple targets (from mixtures of DNA fragments or mRNA molecules) often produces a biased sample, since cDNAs of differing lengths and composition are amplified with differing efficiencies (). The bias is especially conspicuous if amplified RNA is used for preparation of a cDNA library, as the library will provide an inaccurate impression of the abundance and diversity of various transcripts.
Chum-RNA allows cDNA synthesis from a single-cell quantity …
ProtoScript® First Strand cDNA Synthesis Kit | NEB
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA).
First Strand cDNA Synthesis Kit | 69001 - Merck Millipore
Eight replicate cDNA reactions were performed for each input quantity of RNA
Chum-RNA allows cDNA synthesis from a single-cell quantity of mRNA
Extraction of nucleic acid and cDNA synthesis Extraction and purification of RNA, miRNA, DNA, ..
Transcriptor First Strand cDNA Synthesis Kit
The RNA-Seq assays optimized by Otogenetics support the use of low sample quantities for RNA-Seq without sacrificing data quality or reproducibility.Otogenetics now offers direct RNA-seq with polyA cDNA synthesis from low input samples.
Using the Transcriptor First Strand cDNA Synthesis Kit, total RNA ..
An alternative to PCR, for amplifying RNA, is the use of RNA polymerase, which is considered to generate non-biased RNA pools. The most commonly-used technique for RNA amplification is a linear amplification method first developed by Van Gelder, Eberwine and coworkers (,), in which small amount of RNA is primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence located upstream of a polythymidylate region, and then T7 RNA polymerase is used to generate amplified antisense RNA (aRNA) after second-strand cDNA synthesis. This technique and the subsequent improved protocols, with or without combination of PCR (), have allowed genome-wide microarray analysis of gene expression, using a single-cell amount of RNA as a starting material ().
Synthesis cDNA froma single-stranded RNA or DNA primer extension;
Notwithstanding the benefits of RNA polymerase, most cDNA library preparation from single-cell amounts of mRNA is performed using PCR amplification. Indeed, a protocol involving a nested round of cDNA synthesis and in vitro transcription in combination with PCR amplification (,,) successfully reduces the required starting material without significantly reducing the overall sensitivity and fidelity. In contrast, use of RNA polymerase alone still requires one microgram total RNA after two-round amplification of complementary RNA (cRNA) (). This is mainly because the optimum concentration for most of the enzymes used in cDNA library preparation, such as reverse transcriptase (RTase), DNA polymerase I and DNA ligase, is more than 1 μM (). That is, the amount of enzyme exceeds the single-cell amount of mRNA one million-fold. It should be remembered that an enzyme's rate of substrate conversion follows the Michaelis–Menten equation, where the Michaelis constant (Km) is equivalent to the substrate concentration at which the rate of conversion is half of the maximum rate of conversion. Thus, if the Km value of an enzyme is higher than the practical concentration, the rate of substrate conversion becomes very slow.
NZY M-MuLV First-Strand cDNA Synthesis Kit, no ..
qScript cDNA SuperMix is a sensitive and easy-to-use 1-tube reagent for first-strand cDNA synthesis that combines a highly-modified RNAse H+ mutant of M-MLV together with ribonuclease inhibitor protein (RIP) in a rigorously optimized formulation for real-time qPCR applications. The stabilized SuperMix formulation has been rigorously optimized to deliver sensitive, linear assay performance across a spectrum of relative abundance and input RNA (10pg - 1ug). qScript cDNA SuperMix reagent performance is unaffected by repetitive freeze/thaw cycling (>20X), conferring greater ease-of-use and consistent assay performance. Oligo (dT) and random primers are pre-blended in a precise ratio to provide equal representation of 5' and 3'-sequences for accurate gene expression quantification. For gene-specific priming (GSP) or two-step RT-PCR of RNA exceeding 1kb total length, see our qScript Flex cDNA Kit.
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