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High Efficient cDNA synthesis kit with gDNA remover
RT-PCR is a powerful method to detect and synthesize cDNA copies of low-copy-number mRNAs. Two enzymes are used: reverse transcriptase to produce single-stranded cDNA copies, which are then used as templates in an amplification reaction catalyzed by a thermostable DNA polymerase. This protocol describes the traditional method of RT-PCR in which the two synthetic reactions are performed separately and sequentially. For this reason, the method is known as two-step RT-PCR.
Many RTs are available from commercial suppliers. and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. lacks 3´ → 5´ exonuclease activity. is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.
CDNA Synthesis Kits | Life Science Research | Bio-Rad
RT-PCR result after diluting Template RNA
Synthesizes first strand cDNA each at serial dilution(1/10) after extracting total RNA from human cancer cell lines(SNU5) with easy-BLUETM Total RNA Extraction Kit(Cat.
RT-PCR from various template
PCR result by first strand cDNA synthesis after extracting total RNA from virus and cell with Viral Gene-spinTM Viral DNA/RNA Extractio Kit(Cat.
RT2 PreAMP cDNA Synthesis Kit - QIAGEN Online Shop
Total RNA, including miRNAs, was polyadenylated andreverse-transcribed with a poly(T) adapter into cDNAs for real-time PCRusing the miRNA-specific forward primer and the sequence complementaryto the poly(T) adapter as the reverse primer.
In contrast to presenting a stepwisedescription of different platforms, we discuss expression profiling ofmature miRNAs by qPCR in four sequential sections: (1) cDNA synthesis;(2) primer design; (3) detection of amplified products; and (4) datanormalization.
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Protocols for cDNA Synthesis & RT-PCR ..
B: Identify Genes Previously "Absent" with FFPE PreAMP.
RNA extracted from a Human FFPE spleen sample were converted to cDNA with (red bars) or without (blue bars) pre-amplification with RT² PreAMP PCR Master Mix and RT² FFPE PreAMP Primer Mix (Human Cancer PathwayFinder). The unamplified and FFPE PreAMP amplified samples were analyzed on the Human Cancer PathwayFinder RT² Profiler PCR Arrays (PAHS-033), and the threshold cycle values (Ct) were obtained. Shown here are 14 genes that are called "Absent" (Ct>35) in the unamplified sample while these genes are shown to be detectable in the FFPE PreAMP sample.
Typical cDNA Synthesis Protocol;
B. RNA extracted from a FFPE spleen sample were converted to cDNA with or without pre-amplification with RT² PreAmp PCR Master Mix and RT² FFPE PreAMP Primer Mix (Custom). The unamplified and FFPE PreAMP amplified samples were analyzed on a custom PCR array containing 45 genes. Shown here is raw Ct comparison between unamplified and amplified cDNA. Only genes which have Cts lower than 33 in the unamplified cDNA are shown here.
The qScript microRNA cDNA Synthesis Kit is an ..
Figure 1: Increased Positive Call Rate for Genes Extracted from FFPE Samples
A. RNA extracted from a 5 year old Human FFPE spleen sample were converted to cDNA with (red bars) or without (blue bars) pre-amplification using RT² PreAMP PCR Master Mix and RT² FFPE PreAMP Primer Mix (Human Cancer PathwayFinder). The unamplified and FFPE PreAMP amplified samples were analyzed on the Human Cancer PathwayFinder RT² Profiler PCR Arrays (PAHS-033), which contains 84 pathway-specific assays, plus controls, including 5 assays for housekeeping genes. The threshold cycle values (Ct) were obtained. Any genes with a Ct
Toyobo Life Science Department -Products cDNA synthesis kits-
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.
protocol: RNA-seq Library Preparation
Degree of RNA intact in the reaction is very important for high quality cDNA synthesis and it is crucial to keep reagents from contamination such as RNase.
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