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Cat. No.: HTATM-50 (for 50T)

Cat. No.: HTATM-200 (for 200T)

Description

Human Telomerase Activity Assay Kit by Probe qPCR for TERT mRNA, also known as the qPCR Dual Fluorescent Probe Human Telomerase Reverse Transcriptase Subunit Assay Kit or the hTERT qPCR Assay Kit, is a kit that uses dual fluorescent probes to quickly and sensitively detect the expression level of the telomerase catalytic subunit (hTERT) in human cells or tissues, thereby indirectly measuring telomerase activity. This kit is contamination-resistant, containing high-quality UDG enzyme and dUTP in optimized proportions, effectively eliminating false positives or low CT values caused by product contamination during PCR amplification.

Telomerase is a special reverse transcriptase composed of protein and RNA, directly related to the synthesis of telomeres at the ends of eukaryotic chromosomes. The length of telomeres in normal somatic cells shortens with cell division, while enhanced telomerase activity can effectively maintain telomere length. Abnormal telomerase activity can lead to uncontrolled cell proliferation and cancer. Telomerase activity is generally undetectable in normal somatic cells, except for human leukocytes (such as activated B lymphocytes and T lymphocytes), human germ line tissues (adult testes and ovaries, excluding mature sperm and oocytes), and proliferative stem cells. However, telomerase activity is abnormally high in most human cancers, as well as some precancerous and benign tumors, making high-sensitivity rapid detection of telomerase activity crucial for early cancer diagnosis.

Human telomerase primarily consists of three components: human telomerase reverse transcriptase (hTERT), human telomerase RNA (hTER or hTR), and human telomerase-associated proteins. hTERT is considered the key component of telomerase activity, and the mRNA level of hTERT corresponds closely to the overall telomerase activity, allowing the measurement of telomerase activity through hTERT mRNA levels.

Currently, the most common methods for detecting telomerase activity include direct and indirect detection methods. Direct detection methods, such as the Telomerase Repeat Amplification Protocol (TRAP), measure telomerase activity by determining the ability to extend DNA templates through nucleic acid electrophoresis or probe hybridization. These methods require protein extraction, are complex, have low sensitivity, or involve radioactive isotopes, posing safety risks. Indirect detection methods measure the mRNA level of hTERT to indirectly reflect telomerase activity. This simpler method involves RNA extraction, reverse transcription to cDNA, and quantitative PCR, providing higher sensitivity and accuracy.

This kit targets the mRNA of the TERT gene and the GAPDH reference gene for detection, providing optimized primers and probes. Each probe is labeled with a FAM or Cy3 fluorescent group at the 5' end and a BHQ1 or BHQ2 quencher group at the 3' end. Before amplification, the quenching group on the probe suppresses fluorescence due to fluorescence resonance energy transfer (FRET). During PCR, primers and probes anneal to the target genes, and the 5'→3' exonuclease activity of Taq polymerase degrades the probe from the 5' end, separating the fluorescent and quencher groups. The quencher group's suppression effect is eliminated, allowing the fluorescent group to emit light. Each PCR cycle releases more fluorescent groups, with fluorescence intensity proportional to the quantity of newly synthesized target fragments. The real-time amplification curve generated by the qPCR instrument allows for the detection of hTERT expression levels.

The kit includes specific primers for RNA reverse transcription (Specific RT Primers), premix for qPCR (Fast Probe qPCR Mix (2X, UDG)), and specific primer/probe mixes (Primer/Probe Mix). Fast Probe qPCR Mix (2X, UDG) contains Fast Taq DNA Polymerase, UDG enzyme, PCR Buffer, dNTPs, dUTP, stabilizers, and magnesium ions. Additionally, the kit provides a positive control (Positive Control) to ensure the kit's proper function.

UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), catalyzes the hydrolysis of the N-glycosidic bond between uracil (dU) bases and deoxyribose in DNA strands containing uracil, thus releasing free uracil. It is mainly used to eliminate contamination issues from PCR amplification products. The principle behind its contamination prevention is as follows: an appropriate amount of dUTP is added to the PCR reaction to replace dTTP and incorporate dUTP into the DNA, forming PCR amplification products containing dU bases. During subsequent PCR reactions, UDG selectively cleaves single- or double-stranded DNA containing dU, which might be introduced as contaminants from previous PCR amplifications, thus preventing potential contamination from affecting the current PCR amplification.

This kit provides Low ROX and High ROX, making it widely compatible with fluorescence quantitative PCR instruments that either require no ROX or need Low ROX or High ROX as calibration dyes. The role of ROX is to correct fluorescence fluctuations unrelated to PCR, thereby minimizing inter-well variability. Such variability may arise from various factors like pipetting errors and sample evaporation. Different fluorescence quantitative PCR instruments have varying requirements for ROX; please choose High ROX, Low ROX, or no ROX according to your specific instrument when preparing the reaction system.

Components

• Fast Probe qPCR Mix (2X, UDG)
• Specific RT Primers (10X)
• Primer/Probe Mix (10X)
• Positive Control (10X)
• Low ROX (50X)
• High ROX (50X)
• Ultrapure Water