Reverse transcription and QPCR reagents
As the PCR can only be performed on DNA, for RNA Expression profiling the mRNA must first be transcribed into cDNA via a reverse transcription reaction. We highly recommend DNase I (RNase-free) treatment of the total RNA preparation(s) prior to cDNA synthesis step, in order to remove any contaminating DNA from your RNA samples. Reverse transcriptase cDNA preparation can be performed using a number of kits available. During reverse transcription it is important to pay attention to the enzymes and the type of primers used in this process.
We offer sevral kits for cDNA synthesis
Verso™ RT-PCR system This kit is designed for efficient, high-yield synthesis of full-length cDNA from all RNA templates. The unique, highly-sensitive RTase Blend containing RNase inhibitor shows a superior sensitivity and dynamic range at all template concentrations. The 1st Strand Synthesis Buffer is optimised for cDNA synthesis at 42–57°C.
The power of fluorescence is utilized to follow up on the QPCR reaction progress. The fluorophores light emission has to be directly related to the amount of the PCR product in each cycle. If the PCR product is very specific, the common and cost effective fluorophor used is the non-specific intercalating dye SYBR® green I. Once the transcription into cDNA is completed, the Real Time PCR reaction is ready to be performed. Due to the nature of this reaction, it is highly recommended to use a Hot-Start Taq polymerase, which enables a convenient reaction set up at room temperature and dramatically reduces the formation of primer-dimers during the reaction setup.
Thermo Scientific RevertAid First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. The kit uses RevertAid Reverse Transcriptase (RT), a recombinant M-MuLV RT which maintains activity at 42-50°C and is suitable for synthesis of cDNA up to 13 kb. RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA templates from degradation.
The RevertAid First Strand cDNA Synthesis Kit is supplied with both oligo(dT)18 and random hexamer primers. The oligo(dT)18 primer anneals selectively to the poly(A) tail of mRNA. Random hexamer primers do not require the presence of the poly(A) tail, therefore, they can be used for transcription of the 5′-end regions of mRNA or cDNA synthesis of RNA species lacking a poly(A) tail (e.g., microRNAs). Gene-specific primers may also be used with the kit.
Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase is a convenient system optimized for cDNA synthesis in 2-step quantitative RT-PCR (RT-qPCR) applications. It provides a dramatically simplified workflow that combines genomic DNA elimination and cDNA synthesis into one-tube procedure.
The kit contains a novel double-strand specific DNase (dsDNase) engineered to remove contaminating genomic DNA from RNA preps in 2 minutes without damage to quality or quantity of RNA. Highly specific activity towards double-stranded DNA ensures that single-stranded DNA (such as cDNA and primers) is not cleaved and dsDNase treated RNA can be directly added to reverse transcription.
We offer sevral mixes for qPCR
ABgene®’s ABsolute™ Blue SYBR® green I QPCR Mixes are optimised for SYBR® Green I assays and contain all the components necessary to perform Quantitative PCR, with the exception of template and primers. The 2x QPCR SYBR® Green Mix contains optimal levels of active SYBR® Green I dye and Thermo-Start® DNA Polymerase supplied in a proprietary reaction buffer that enables detection of low copy number targets. The kits are available with the option of dUTP and/or ROX or Fluorescein reference dyes.
If a higher specificity or a multiplex QPCR is required, the reaction can be performed using specific probes, such as dual labeled probes. The cost per reaction is somewhat higher than SYBR® green I reaction, because one must order the specific dual labeled probe. For dual labeled probes QPCR, we offer ABgene®’s ABsolute™ Blue QPCR Mixes which are optimised for use with hybridization probes and contain all the components necessary to perform quantitative PCR, with the exception of template and primers/probes. The 2x QPCR Mix contains a proprietary reaction buffer that provides high sensitivity, specificity and reproducibility. The mix is also optimized for end-point applications, including SNP assays. All kits are available with the option of dUTP and/or ROX passive reference dye.
Thermo Scientific Luminaris Color HiGreen and Luminaris HiGreen qPCR Master Mixes are universal ready-to-use solutions optimized for qPCR and two-step RT-qPCR. The master mixes include Thermo Scientific Hot Start Taq DNA polymerase, uracil-DNA glycosylase (UDG) and dNTPs in an optimized PCR buffer. Only the template and primers need to be added. The SYBR Green I intercalating dye allows for DNA detection and analysis without using sequence-specific probes.
The Hot Start Taq DNA polymerase in combination with an optimized buffer ensures PCR specificity and sensitivity. dUTP and UDG are included in the mix for carry-over contamination control (see Reference 1). The Luminaris Color HiGreen qPCR Master Mixes are supplemented with an inert blue dye and a separate Yellow Sample Buffer that contains a yellow dye. The qPCR reaction mix containing both components is green.
Tamar offers an extensive range of reagents, plastics, custom made TaqMan® and FRET probes and other custom made modified oligos as well as kits for Real-Time PCR. As always we supply top quality and service at a very competitive price, specifically to meet your needs in modern life science research and diagnostics.
TaqMan® is a registered trademark of Roche Molecular Systems, Inc.
SYBR® is a registered trademark of Molecular Probes