cleanamptm primers for improved hot start pcr  (TriLink)

Journal: BMC Molecular Biology

Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

doi: 10.1186/1471-2199-10-113

Figure Lengend Snippet: One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

Article Snippet: Gene-specific PCR primers for the PBGD, ABCA5, ABCA6, ABCA7, ABCC10 genes were prepared as unmodified and as CleanAmpTM Precision (TriLink BioTechnologies, Inc.), which contains two thermolabile phosphotriester modifications at the 3'-terminal phosphodiester internucleotide linkage and the penultimate phosphodiester internucleotide linkage [ - ].

Techniques: One Step RT-PCR, Reverse Transcription

Journal: BMC Molecular Biology

Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

doi: 10.1186/1471-2199-10-113

Figure Lengend Snippet: Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

Article Snippet: Gene-specific PCR primers for the PBGD, ABCA5, ABCA6, ABCA7, ABCC10 genes were prepared as unmodified and as CleanAmpTM Precision (TriLink BioTechnologies, Inc.), which contains two thermolabile phosphotriester modifications at the 3'-terminal phosphodiester internucleotide linkage and the penultimate phosphodiester internucleotide linkage [ - ].

Techniques: One Step RT-PCR, Amplification, Incubation

Journal: BMC Molecular Biology

Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

doi: 10.1186/1471-2199-10-113

Figure Lengend Snippet: Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

Article Snippet: Gene-specific PCR primers for the PBGD, ABCA5, ABCA6, ABCA7, ABCC10 genes were prepared as unmodified and as CleanAmpTM Precision (TriLink BioTechnologies, Inc.), which contains two thermolabile phosphotriester modifications at the 3'-terminal phosphodiester internucleotide linkage and the penultimate phosphodiester internucleotide linkage [ - ].

Techniques: One Step RT-PCR, Reverse Transcription, Amplification, Standard Deviation, Agarose Gel Electrophoresis

Journal: BMC Molecular Biology

Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

doi: 10.1186/1471-2199-10-113

Figure Lengend Snippet: Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

Article Snippet: Gene-specific PCR primers for the PBGD, ABCA5, ABCA6, ABCA7, ABCC10 genes were prepared as unmodified and as CleanAmpTM Precision (TriLink BioTechnologies, Inc.), which contains two thermolabile phosphotriester modifications at the 3'-terminal phosphodiester internucleotide linkage and the penultimate phosphodiester internucleotide linkage [ - ].

Techniques: One Step RT-PCR, RNA Expression, cDNA Synthesis

Journal: BMC Molecular Biology

Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

doi: 10.1186/1471-2199-10-113

Figure Lengend Snippet: PCR Primer and hydrolysis probe sequences for the five human total RNA targets.

Article Snippet: Gene-specific PCR primers for the PBGD, ABCA5, ABCA6, ABCA7, ABCC10 genes were prepared as unmodified and as CleanAmpTM Precision (TriLink BioTechnologies, Inc.), which contains two thermolabile phosphotriester modifications at the 3'-terminal phosphodiester internucleotide linkage and the penultimate phosphodiester internucleotide linkage [ - ].

Techniques: Sequencing