Genomic DNA was extracted from the primary tumours and noncancerous mucosa using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). TL was measured using a quantitative (Q)-PCR assay kit (ScienCell Research Laboratories, Carlsbad, CA, USA). Genomic DNA (10 ng) was amplified with a TB Green Premix Ex Taq II system (Takara, Tokyo, Japan) using a Takara Thermal Cycler Dice Real Time System TP8000. The data analysis was conducted according to the manufacturer's instructions. For each DNA sample, two consecutive reactions were performed: the first to amplify a single-copy reference (SCR) gene and the second for the telomere sequence. The SCR primer set recognises and amplifies a 100 bp-long region on human chromosome 17 and serves as a reference for data normalisation. The Q-PCR conditions were as follows: 95 °C for 10 min followed by 32 cycles of 95 °C for 20 s, 52 °C for 20 s, and 72 °C for 45 s. All reactions were performed in triplicate. After Q-PCR was performed, we used the instrument's analysis software to analyse the data 12 (link).
Qpcr assay kit
Manufactured by ScienCell
Sourced in United States
The QPCR assay kit is a laboratory product used to perform quantitative polymerase chain reaction (qPCR) analysis. The kit contains the necessary reagents and components to quantify the amount of a specific nucleic acid sequence in a sample.
Automatically generated - may contain errors
4 protocols using qpcr assay kit
1
Quantitative Telomere Length Measurement
2
Telomere Length Quantification in Hippocampi
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The hippocampi were dissected from brain and applied to genomic DNA extraction according to the kit’s protocol (Qiagen, Hilden, Germany). The telomere length was assayed with qPCR Assay kit (ScienCell, M8918, Carlsbad, CA) and qPCR master mix (Roche, #06402712001, Basel, Switzerland). Briefly, 2 ng genomic DNA template was well mixed with telomere (Tel) or single copy reference (SCR) primer and qPCR master mix in 20 μl reaction volume. The processing for qPCR program was as follows: Initial denaturation 95 °C for 10 min, denaturation 95 °C for 20 seconds, annealing 52 °C for 20 seconds, and extension 72 °C for 45 seconds. The total number of cycles was 32. Relative telomere length of the target sample to the reference sample was expressed by fold, which is equal to 2-(ΔCq Tel− ΔCqSCR).
3
Quantifying Relative Telomere Length in HIV Patients
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We used the relative human telomere length quantification qPCR assay kit from ScienCell Inc. (RHTLQ cat # 8908, ScienCell Inc., Carlsbad, CA, USA) for TL analysis. QPCR data was analyzed using the Comparative CT method, where relative telomere length was calculated based on the formula T/S = 2−ΔΔCt, where ΔCt = Ct telomere-Ct SCR or (single copy reference) primer. The relative telomere length was calculated for the samples from patients on cART vs. patients who were cART naïve, and also the cDNA isolated from HTHU vs. the HTHU/HIV cells.
4
Quantifying Telomerase Activity by qPCR
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Telomerase is a ribonucleic acid-protein complex composed of a single long non-coding RNA, called telomerase RNA, and associated proteins. We used a telomerase activity quanti cation qPCR assay kit (ScienCell, Carlsbad, CA, USA #8928) to measure the products of telomerase activity that are ampli ed by qPCR. The cell lysis buffer enables the release of telomerase in the native state and the telomere primer set (TPS) recognizes and ampli es newly synthesized telomere sequences in the assay. Cell proteins were extracted by cell lysis buffer supplemented with PMSF 0.1 M in isopropanol and β-mercaptoethanol.
After cell protein extraction telomerase reaction was performed as described in the product protocol. First, the telomerase reactions (including 0.5 µL cell lysate sample, 4 µL 5×g telomerase reaction buffer and 15.5 µL nuclease-free H 2 O) were incubated at 37 °C for 3 h. The reaction was stopped by heating at 85 °C for 10 min and the reaction tubes were centrifuged at 1,500 ×g for 10 s. Finally, qPCR was performed to analyze the telomer production by telomerase. The qPCR process was 10 min at 95 °C, followed by 40 cycles (denaturation, 95 °C for 20 s; annealing, 95 °C for 20 s and extension, 72 °C for 45 s).
After cell protein extraction telomerase reaction was performed as described in the product protocol. First, the telomerase reactions (including 0.5 µL cell lysate sample, 4 µL 5×g telomerase reaction buffer and 15.5 µL nuclease-free H 2 O) were incubated at 37 °C for 3 h. The reaction was stopped by heating at 85 °C for 10 min and the reaction tubes were centrifuged at 1,500 ×g for 10 s. Finally, qPCR was performed to analyze the telomer production by telomerase. The qPCR process was 10 min at 95 °C, followed by 40 cycles (denaturation, 95 °C for 20 s; annealing, 95 °C for 20 s and extension, 72 °C for 45 s).
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